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 Difficulty Time Required Very Long (1+ months) Prerequisites Previous experience using a multimeter and being familiar with the physics of electricity is helpful, but not required. Material Availability The microbial fuel cell kit needs to be special-ordered from the Science Buddies Store. Cost Average ($50 -$100) Safety Be sure to wear the gloves supplied with the kit when handling the microbial fuel cell's electrodes (its cathode and anode). The electrodes are made of a conductive material called graphite fiber and should not be placed near electronics or power plugs, or have their fibers dispersed in the air. The fibers will cause electrical shortages when they come in contact with electronics. Wear gloves when working with human urine. Adult supervision is recommended.

## Abstract

Every day, we produce a lot of sewage (wastewater full of feces and urine). In fact, it adds up to 6.4 trillion liters of urine alone produced worldwide each year! The sewage is collected and then treated or disposed of. But what if, along the way, there were a way to make that sewage do something useful? Human urine is rich in nutrients, and some bacteria actually thrive on eating those nutrients. There are also devices called microbial fuel cells that can generate electrical power by using certain bacteria. Could human urine be used to generate electricity in a microbial fuel cell? Find out for yourself in this science project.

## Objective

Investigate how adding different amounts of urine to a microbial fuel cell changes its power output.

## Credits

Teisha Rowland, PhD, Science Buddies
Svenja Lohner, PhD, Science Buddies

Thanks to Bob Rowland of ColdQuanta Inc., for assistance with testing this project idea, and to Ben Finio, PhD and Howard Eglowstein of Science Buddies, and Keegan Cooke, Executive Director at Keego Technologies LLC, for their feedback.

### MLA Style

Science Buddies Staff. "Powered by Pee: Using Urine in a Microbial Fuel Cell" Science Buddies. Science Buddies, 28 Nov. 2016. Web. 28 Feb. 2017 <http://www.sciencebuddies.org/science-fair-projects/project_ideas/EnvSci_p061/environmental-science/microbial-fuel-cell-urine.shtml?from=Blog>

### APA Style

Science Buddies Staff. (2016, November 28). Powered by Pee: Using Urine in a Microbial Fuel Cell. Retrieved February 28, 2017 from http://www.sciencebuddies.org/science-fair-projects/project_ideas/EnvSci_p061/environmental-science/microbial-fuel-cell-urine.shtml?from=Blog

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Last edit date: 2016-11-28

## Introduction

Each year, people produce about 6.4 trillion liters of urine worldwide! That is a lot of waste that needs to be collected and then properly treated and/or disposed of. But maybe we do not need to see it as just "waste;" it might be able to serve a useful purpose while it is being processed. Urine is full of nutrients, primarily nitrogen (in the form of urea), along with other compounds, including chloride, sodium, potassium, and creatinine. Because of this, human urine has sometimes been used as a fertilizer for plants. (For more on this, see the Science Buddies science project idea Growing Great Gardens: Using Human Urine as a Fertilizer.)

Among other uses, the nutrients in urine can also be used to feed microbes (microscopic organisms), like bacteria. Why would this be useful? As it turns out, in the early 1900s, scientists showed that microbes could make electricity, which is the basis of microbial fuel cell (or MFC) technology. As natural resources are being depleted, scientists' attention has shifted to pursuing alternative energy sources, such as MFCs, even more than before.

A microbial fuel cell, also known as a biological fuel cell, is a device that can use microbial interactions to generate electricity. It is a renewable, clean source of energy, making it quite appealing. An MFC has an anode, a cathode, and an area that separates the two (called a membrane). Anodes and cathodes are both electrodes. An electrode is something that conducts electricity, with electricity either flowing into, or out of it. Specifically, an anode has electricity flowing into it, whereas a cathode has electricity flowing out of it. So, for an MFC to function, electricity must be made to flow into the anode and then leave from the cathode. How is this accomplished?

To answer this question, we will look at MFCs that use microbes from the soil to generate electricity. When you think of electricity, and how it can be made in nature, you may think of lightning and electric eels, though you probably do not think about microbes! But some types of soil bacteria can help generate electricity, too. These bacteria, known as electrogenic bacteria, include the Shewanella species, which can be found in almost any soil on Earth and are shown in Figure 1, and the Geobacter species, which prefer living in soil deep underground or even under the ocean, where no oxygen is present. How can these bacteria help make electricity? The soil bacteria eat what is in the soil, such as microscopic nutrients and sugars, and in turn, produce electrons that are released back into the soil. Electrons are subatomic particles that have a negative electric charge. These electrons can be harnessed and used to create electricity, which is a form of energy. For example, if a lightbulb has enough electricity flowing through it in the correct way, the lightbulb will light up.

Figure 1. This is a high-magnification image of Shewanella bacteria, specifically the species S. oneidensis. The bacteria are the cylindrical rods scattered in this image. (The other parts of the image are ice pieces that the bacteria were submerged in to take this picture.) (Image credit: PLoS Biology)

In an MFC using these soil bacteria, the anode is buried in the damp soil. Down there, the bacteria multiply and cover the anode (creating a biofilm on it), supplying it with a lot of electrons. At the same time, electrons are taken away from the cathode. How does this happen? While the anode is buried in the soil, the cathode sits on top of the soil, leaving one of its sides completely exposed to the air. Electrons from the anode travel up a wire to the cathode and, once there, they react with oxygen (from the air) and hydrogen (produced by the bacteria as it digests the nutrients in the soil) to create water. The anode is buried deep enough, where there is no oxygen, so this reaction could not take place right next to the anode. See Figure 2 for a visualization of this process. The more electron-producing, soil-munching bacteria are in the soil, the more electricity the MFC produces.

Figure 2. This diagram shows the reactions taking place in a microbial fuel cell (or MFC) that make it generate electricity. (Wikimedia Commons, 2010, MFCGuy2010)

To evaluate the overall performance of an MFC, usually its power output is determined. This is done by measuring the voltage across a fixed resistor that you attach to the MFC and from that, power is calculated using a derivation of Ohm's law, as shown in Equation 1.

Equation 1:

• P is the power in watts (W),
• V is the voltage (V), and
• R is the resistance in ohms (Ω).

If you use several different resistors, you can generate a power-resistance curve, which allows you to determine the maximum power output of your microbial fuel cell, as explained in more detail in the Procedure. As the bacteria will start generating power from the food present in the soil, the power output will start increasing over time and eventually reach a steady-state.

In this environmental science project, you will investigate how adding human urine to an MFC changes its electrical power output. Are you shocked that you will be using human urine? Do not be—even NASA has experimented with using urine as a fertilizer! Urine is actually relatively clean. In fact, human urine in the bladders of individuals without bladder and kidney infections is sterile. At the beginning of urination, the flow takes with it any bacteria in the urethra, cleaning the urethra, but contaminating the urine. While this means the initial flow might not be sterile, the mid-flow urine will be sterile. And as we mentioned, human urine is continually being made in large amounts, making it a renewable resource, and it is rich in nutrients (mainly nitrogen) that bacteria like Shewanella and Geobacter species can eat. And the more Shewanella and Geobacter bacteria that are in the soil of an MFC, the more electricity it makes. How do you think adding urine to an MFC will affect its power output? Do you think a certain amount needs to be added for the power output to increase? Is it possible to add too much and cause the bacteria to die? There are all sorts of interesting questions you can ask in this science project; get ready to figure some of the answers out!

## Terms and Concepts

• Urine
• Nitrogen
• Microbes
• Bacteria
• Alternative energy sources
• Microbial fuel cell (MFC)
• Renewable
• Electrode
• Shewanella
• Geobacter
• Electrons
• Electricity
• Power output
• Resistor
• Ohm's law
• Watt (W)
• Voltage (V)
• Ohm (Ω)
• Sterile

### Questions

• What nutrients can bacteria eat in human urine?
• How does an MFC work?
• How do soil bacteria help make electricity in an MFC?
• How do you think adding a small amount of human urine to the MFC will affect its power output? How about adding a much larger amount of urine?

## Bibliography

These resources will give you more information about microbial fuel cells and using waste to create power:

## News Feed on This Topic

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Note: A computerized matching algorithm suggests the above articles. It's not as smart as you are, and it may occasionally give humorous, ridiculous, or even annoying results! Learn more about the News Feed

## Materials and Equipment

These specialty items can be purchased from the Science Buddies Store:

• Microbial Fuel Cell kit (1). Includes:
• Microbial Fuel Cell vessels (2)
• Anodes (2)
• Cathodes (2)
• Hacker boards (2)
• Capacitors (2)
• LEDs (2)
• Digital clock/thermometer to be powered by the microbial fuel cell (1)
• Set of seven resistors
• Alligator clips and jumper wires (2)
• Digital multimeter
• Nitrile gloves (1 pair)

You will also need to gather these items (not included in the kit):

• Optional: Old newspapers to protect your work area
• Soil (at least 4 cups) or about 400 g of sifted soil
• Soil from just about anywhere works, such as a backyard, park, open space, or even a riverbed. Just make sure the soil has not been treated with pesticides and that you have permission to take some of it.
• Soil can also be purchased from a plant nursery, a hardware store, or online. When you buy your soil, choose organic topsoil that has not been sterilized or treated with pesticides. Do not use soils with little white foam balls, vermiculite pieces, or perlite, that are used to aerate the soil and avoid soils with peat moss.
• A sieve, plastic strainer or colander to remove large particles from the soil
• Large mixing bowl that can hold all the soil
• Measuring cups or 100 mL graduated cylinder. A 100 mL graduated cylinder is available from Amazon.com
• Distilled water (at least 250 mL). This can be purchased at most grocery stores.
• Paper towel or rag
• Permanent marker
• Stopwatch
• Human urine; do not collect urine from anyone known to have a bladder, kidney, or urinary tract infection.
• Clean glass jar with lid, approximately 25 oz. Make sure that the jar and lid are completely cleaned and dried.
• Medicine dropper or transfer pipettes, available from Amazon.com
• Lab notebook

Disclaimer: Science Buddies occasionally provides information (such as part numbers, supplier names, and supplier weblinks) to assist our users in locating specialty items for individual projects. The information is provided solely as a convenience to our users. We do our best to make sure that part numbers and descriptions are accurate when first listed. However, since part numbers do change as items are obsoleted or improved, please send us an email if you run across any parts that are no longer available. We also do our best to make sure that any listed supplier provides prompt, courteous service. Science Buddies does participate in affiliate programs with Amazon.com, Carolina Biological, Jameco Electronics, and AquaPhoenix Education. Proceeds from the affiliate programs help support Science Buddies, a 501(c)(3) public charity. If you have any comments (positive or negative) related to purchases you've made for science fair projects from recommendations on our site, please let us know. Write to us at scibuddy@sciencebuddies.org.

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## Experimental Procedure

Microbial Fuel Cells in Science Fairs

Working with microbial fuel cells involves growing soil bacteria. Because of this, many science fairs, including those associated with the International Science and Engineering Fair (ISEF) have requirements which need to be met before you start your project. We recommend you:

• Check with your teacher or science fair coordinator about any requirements
• Read the Science Buddies Microorganisms Safety Guide to learn how to safely handle bacteria
• Fill out a Risk Assessment Form (required by ISEF affiliated science fairs for all MFC projects)

### Setting up the Microbial Fuel Cells

You will assemble two identical microbial fuel cells that contain the same soil material. Later in the procedure you will add a different amount of urine to each one of them and compare their power outputs. Do you think more urine will result in more power?

1. First, watch the video or follow the step-by-step instructions to see how to assemble the microbial fuel cells.
1. Place a plastic strainer or colander over a large mixing bowl.
2. Measure a total of about 2 cups (about 500 milliliters [mL]) of your soil for each of the microbial fuel cells into the strainer (4 cups in total). Gently shake the strainer over the bowl so that the soil is strained and any small, hard particles (such as rocks, pebbles, twigs, etcetera) are removed from the soil. You will likely need to be patient; it may take several minutes to strain the soil. When you are done, you should have about 400 g of fine, sifted soil in the bowl.
1. It is important to remove these particles from the soil because they can aerate the soil and inhibit the desired bacteria from growing (the bacteria do not want to be exposed to oxygen).
3. Add distilled water and mix it in until your topsoil mud feels like cookie dough. Add more water if the mud is too crumbly, or add more topsoil if the mixture feels too wet.
4. When you have prepared your topsoil mud, set it aside and wash your hands.
2. Carefully take the MFC pieces out of the box and lay them out. Identify the different components and use masking tape and a permanent marker to label one of the devices with "1 mL urine" and the other one with "5 mL urine".
3. Put on the gloves that came with the MFCs and start assembling the first microbial fuel cell.
4. Take out the green and orange wires that came with the MFC electrodes. Bend each wire where the plastic part ends so that each wire is now at a 90° angle (shaped like a capital "L"), as shown in the video above.
5. Remove the MFC anode from its bag. (The anode is the thinner, black, felt-like circle.)
1. Safety Note: The MFC's cathode and anode (its electrodes) are made of a conductive material called graphite fiber. Do not put the cathode or anode near electronics or power plugs, and do not disperse the fibers in the air, as the fibers will cause electrical shortages when in contact with electronics.
6. Straighten the metal part of the green wire and carefully insert it into the anode, as shown in Figure 3. Make sure the wire goes straight and does not poke out on the top or bottom sides of the anode.

Figure 3. Insert the metal part of the green wire into the anode circle.
1. Repeat steps 6 and 7 using the cathode (the thicker, black, felt-like circle) and the orange wire (which is shorter than the green wire).
2. Take the soil mud that you prepared in step 2 and use it to fill the first vessel up to the line next to the "1" on the plastic vessel (marking 1 centimeter [cm]). Once filled, pat the mud so that its surface is smooth.
1. Tip: You may want to cover the surface you are working on with old newspapers to prevent mud from getting on it.
2. When you are finished, rinse the mud off your gloves and dry them (but do not take them off yet).
3. Put the anode on top of the mud in the vessel, as shown in Figure 4.
1. The green wire from the anode should be sticking up. The green wire should not be stuck down in the mud.
2. Gently press the anode flat against the mud so that no air bubbles are under the anode. Note: Removing the air bubbles is important, as the trapped oxygen can prevent the formation of an anaerobic bacterial biofilm and reduce the power output of your microbial fuel cell.

Figure 4. Place the anode on top of the 1 cm of mud.
1. Use more soil mud to fill the vessel up to the line next to the "5" mark (marking 5 cm). Once filled, again pat the mud so that its surface is smooth.
1. Run the green wire along the side of the vessel.
2. Rinse the mud off your gloves and dry them.
2. Gently place the cathode on top of the mud and press it as flat as you can, as shown in Figure 5.
1. The orange wire from the cathode should be sticking out of the top side.
2. Do not let any mud or liquid cover the top of the cathode.
3. It is best to arrange the cathode so that its orange wire is about 1–2 cm to the left of the green wire.
4. Let the mud rest in the vessel for a few minutes. Then carefully pour off any excess liquid.

Figure 5. Add the cathode on top of the 5 cm of mud.
1. Use a clean paper towel or rag to wipe any mud off the vessel's rim. Then take off your gloves.
2. Take the white plastic lid and pass the wires through the small holes in the lid. Arrange the wires so that the orange wire is on the left and the green wire is on the right when the semicircular indentation on the lid is facing the front. Then carefully snap the lid onto the plastic vessel.
3. Take out the hacker board (the small green circuit). Attach it into the lid's rectangular indentation.
4. Locate the "+" and "-" ports (the holes) on the hacker board. Plug the cathode's wire (orange) into the "+" port and the anode's wire (green) into the "-" port.
5. Locate ports 1 and 2 on the hacker board. Plug the blue capacitor (the small, cylindrical item with two longer metal prongs) into these ports. The blue capacitor's longer prong should go into port 1 and the shorter prong into port 2. Note: The orientation of the capacitor is important. If you reverse the short and long prongs, it will not work and might even damage the capacitor. You may need to bend the capacitor's longer end slightly so that the capacitor's prongs fit into the ports well.
6. Plug the red LED below the capacitor into ports 5 and 6. (Ports 3 and 4 will remain empty.) The LED's longer prong should go into port 5 and the shorter prong into port 6. Note: The LED will only work if inserted in this orientation. If you accidentally reverse the prongs, the LED will not light up. You may need to bend the LED's longer end slightly so that the LED's prongs fit into the ports well.
7. Make sure that the wires, capacitor, and LED are all securely in place. The assembled hacker board on the top of the MFC should now look like Figure 6.

Figure 6. When you have finished assembling your MFC and its hacker board, the top should look like the one in this image.
1. After assembling the first MFC, set up the second microbial fuel cell using the rest of the prepared soil from step 2.
2. Once both microbial fuel cells are assembled, set the MFCs indoors, at normal room temperature (about 19–25° Celsius [C], or 66–77° Fahrenheit [F]), in a place where they will not be disturbed. The MFCs should remain in the same location the entire time after you set them up because if they are moved this could disrupt the growth of the bacteria. It should take 3–7 days before the red LEDs on the hacker board start blinking, but you will start taking measurements before that, as described in the next section. Note: Temperature variations can cause changes in the power output of the microbial fuel cells due to different bacteria activities.

### Measuring Power Output and Adding Urine

You will measure the power output for both of your microbial fuel cells every day. Once the power output seems to have stabilized, you will add a different amount of urine to each one of the microbial fuel cells. The urine should change the power output of the MFC. How do you think the MFC's power output will be affected?

1. One day after setting up your microbial fuel cells, check to see if the LEDs are blinking. Most likely, they will not be, but check to make sure. Watch the LEDs for 2 minutes to see if they are blinking.
1. If one or both of the LEDs are blinking, time how many seconds apart the blinks for each one are.
1. To do this, start a stopwatch as soon as you see the LED blink and stop the stopwatch when the LED blinks again.
2. If the LED is blinking faster than once every 5 seconds, do not time the seconds between blinks, but instead time the blinks per second. Time a 10-second interval and count how many times the LED blinks in this period and then divide this by 10 to get blinks per second.
3. Repeat step 1.a.i. or 1.a.ii. two more times so you have a total of three counts.
4. Record your results for each MFC in your lab notebook in a data table like Table 1. (If you counted blinks per second, as in step 1.a.ii., change the heading from "Seconds Between Blinks" to "Blinks per Second.") Calculate the average for your three counts and record that, too.

Day Count #1 Count #2 Count #3 Average
1
2
3
Table 1. Each day, check both MFCs to see if the LEDs are blinking. If they are, record how many seconds elapse between the blinks (or how many blinks there are per second) for each, making three separate counts. Record the results for each microbial fuel cell in a data table like this one in your lab notebook.

1. Next, measure the power output for both of the MFCs using the multimeter that comes with the Microbial Fuel Cell kit. You can either watch the video below or follow the step-by-step instructions. If you need help using a multimeter, consult the Science Buddies reference How to Use a Multimeter, as well as the instructions that came with your multimeter.

1. To measure the MFC's power output, remove the capacitor and LED from the hacker board. Then remove the orange wire from the "+" port and plug it into port 3. This means that the orange wire should be in port 3, the green wire should still be in the "-" port, and all other ports should be empty.
2. Place a resistor between ports 5 and 6, as shown in the video above. For the resistors the orientation does not matter.
1. Several resistors come in the MFC kit. Start with the largest-capacity resistor, which will be a 4.7 kΩ resistor. (Ω, the capital Greek letter Omega, is the symbol for ohms, the unit used to measure resistance. 1 kilo-ohm, or 1 kΩ, is 1000 ohms.)
2. Resistors' values are labeled using color-coded bands. Use Figure 7 to determine the resistance for each resistor.
1. Tip: If you want, you can confirm the resistance of any resistor using your multimeter by setting it to measure resistance (usually a "Ω" symbol for ohms) and connecting the multimeter's leads on the wire ends of the resistor.

Figure 7. Use this resistor color chart to determine the resistance for each resistor.

3. Leave the resistor plugged in for 5 minutes.
3. After the resistor has been plugged in for 5 minutes, use your multimeter to measure the voltage across the resistor.
1. Make sure the multimeter's black wire is plugged into the "COM" port and its red wire is plugged into the "VΩMA" port on the multimeter.
2. Set the multimeter to measure DC voltage. This is marked as "V" with a straight line next to it. Specifically turn the dial to "2000 m."
3. Clip the multimeter's red lead to the resistor's metal wire that is plugged into port 5. Then clip the multimeter's black lead to the resistor's metal wire that is plugged into port 6, as shown in the video. Read the multimeter's screen to see what the voltage is (in millivolts [mV]).
4. If the voltage seems to be changing a little, such as decreasing slightly over the period of a few seconds, watch the readings on the multimeter for a few seconds more until they stabilize (and stay the same for a few seconds). Use the stabilized value.
1. If the readings are still changing after several seconds, or if your readings are 0 mV, make sure all of the wires are correctly and securely plugged into the circuit (both the cathode and anode wires, and the resistor's wires), disconnect the multimeter's leads from the resistor, and come back in another 5 minutes. Then repeat step 2.c.
5. Record your results for both of the microbial fuel cells in your lab notebook in a data table like Table 2.

Day and Time:
Resistance (ohms) Voltage (mV) Power (μW)
4700
2200
1000
470
220
100
47
Table 2. In your lab notebook each day, create a data table like this one for each microbial fuel cell to record your voltage measurements. Do not forget to write down the date and the time you started taking measurements on the top line.

4. Disconnect the multimeter's clips from the resistor. Remove the resistor.
5. Repeat steps 2.b.–2.d. until you have tested the MFC with all of the resistors in the kit. Start with the resistor with the largest resistance value and end with the resistor with the smallest resistance value.
2. Once you have finished taking your voltage measurements for the first MFC, plug the wires, the capacitor, and LED back into the hacker board, as described in steps 16–18 in the Setting Up the Microbial Fuel Cells section and repeat the voltage sweep (step 2 and 3) for the second microbial fuel cell.
3. Calculate the power output (in microwatts, or µW) for each resistor and both microbial fuel cells. You can calculate this by using a derivation of Ohm's law, as described in the Introduction, Equation 1.
1. Note: It is important to convert the voltage measurements into power output measurements. The power output depends on the resistors you use, so you cannot determine how well the MFC is performing by just looking at the voltage measurements alone; they need to be converted into power for them to be meaningful.
2. To use Equation 1, you will need to convert your voltage readings from millivolts (mV) to volts (V). To do this, divide the millivolt values by 1000 to give you volts.
1. For example, if you had a voltage reading of 45 mV, this would equal 0.045 V.
4. Once you have calculated it, record the power for each resistor in the data table (like Table 2) in your lab notebook.
4. Determine what the peak power of your MFCs is.
1. In your data tables for both microbial fuel cells, look at the power produced using each resistor. The peak power is the highest power produced by any of the resistors.
2. If you want to visualize this, you can plot your data for the day on a graph, putting the resistance of the resistors on the x-axis (horizontal axis) and the power on the y-axis (vertical axis) for both MFCs. A sample graph is shown in Figure 8.
1. You should see a curve, with the peak power at the top of the curve, as shown in the sample graph.
3. Make a note in your lab notebook of what the peak power for both MFCs is each day, by circling or highlighting this value in your data table.
4. Although it will not be explored in this science project, you might be interested to know that the peak power tells you what the internal resistance of your MFC is. The resistor that gives the peak power is closest to the internal resistance of the MFC. This may change a little over time.

Figure 8. This sample graph shows possible power output data using the resistors with the MFC. In this sample, the peak power was found using a 1 kΩ resistor, and the peak power is about 45.5 µW.
1. Repeat steps 1–5 each day until it looks like the power output (the peak power) for both of your microbial fuel cells is stabilizing.
1. Take these measurements around the same time every day. This will limit variables affecting your results (such as changes in temperature).
2. For step 1, it should take 3–7 days for the LEDs to start blinking. However, even if the LEDs never blink, you may still be able to do this science project; be sure to continue to take the power output measurements every day.
1. Tip: See the Frequently Asked Questions section for what to do if the LEDs do not blink, or if they were blinking and unexpectedly stopped blinking.
3. For step 2, you should see the power output slowly increase.
1. For each day, make a data table like Table 2 for each MFC in your lab notebook to record your results and use them to determine the peak power.
4. After about 7–14 days, the power output should stabilize.
1. It may stabilize anywhere between 10 μW to 200 μW or more. A lot depends on the topsoil you are using and other factors. Wherever it stabilizes, it should make enough power to blink the LED at least once every 30 seconds.
1. Tip: If the power output seems low, see the Frequently Asked Questions section for suggestions on what to check and try.
2. When it stabilizes, the peak power should not change by more than about 10% for at least three days in a row.
1. Do not worry if your peak power changes by a little more than this. If it has been at least 14 days and when you graph the peak power (as described in step 6.d.iii., following) it looks like it is stabilizing (meaning it is not steadily increasing or steadily decreasing from day to day), then it has probably stabilized enough.
2. Keeping this in mind, if it still does not look like your peak power is stabilizing, see the Frequently Asked Questions section for suggestions on what to check and try.
3. Tip: Making graphs of your data as you collect it for both MFCs may help you see if the power output is stabilizing. If you do this, put the date on the x-axis and the power output (peak power) for each day on the y-axis. Does the peak power appear to be stabilizing?
4. The time between LED blinks should also stabilize.
2. Once it appears that the power output has stabilized in both MFCs, it is time to carefully open up both of the microbial fuel cells and add 1 mL of urine to one MFC and 5 mL to the other, one drop at a time.
1. First, harvest your urine. This step is similar to collecting a urine sample at the doctor's office.
1. Make sure the glass jar and its lid are clean. Be sure to rinse the jar thoroughly with water (to get rid of any antibacterial soap residue that could harm the soil bacteria) and then let it dry before using it to collect urine.
2. Urinate into a toilet for a few seconds (to get rid of any contaminated urine) and then capture the rest of the urine in a clean, approximately 25 oz. glass jar. The mid-flow urine should be sterile. See the Introduction for an explanation of why. When you are done, cover the jar with the lid.
1. Note: If you need to clean any spills on the outside of the jar, be sure the lid is on tight before using any cleaning solutions so they do not come into contact with the urine sample in the jar and potentially harm the soil bacteria in the MFC.
3. You can use the urine for up to one week if it is stored in a refrigerator. Be sure it is clearly labeled and kept away from food. When you want to add the urine to the MFCs, set it out at room temperature for 2–3 hours to allow it to reach room temperature.
2. Take your measurements for the day, as usual, before starting the treatments.
3. Then, unplug the anode and cathode from the hacker board and carefully remove the lid of the first microbial fuel cell labeled "1 mL urine".
4. Put your gloves on and gently lift up the cathode, being careful not to get any mud on top of the cathode.
1. Safety Note: Remember, the MFC's electrodes are made of a conductive material called graphite fiber. Do not put the cathode near electronics or power plugs, and do not disperse the fibers in the air, as the fibers will cause electrical shortages when they come in contact with electronics.
5. Use a clean medicine dropper or transfer pipette to suck up 1 mL urine from the jar. Add the urine to the top of the mud one drop at a time and spread it out evenly across the mud's surface. Note: 1 mL of urine equals approximately 20 drops.
6. Wait 5 minutes to let the urine soak into the mud a little. (If you immediately put the cathode back on the mud, the cathode will likely soak up much of the urine). Flatten the mud surface with a clean spoon again before you re-assemble the MFC.
7. Then assemble the MFC exactly as you put it together before, following the instructions from the "Setting Up the Microbial Fuel Cells" section to make sure that the wires are twisted together properly and everything is reconnected to the hacker board correctly.
1. Specifically, follow steps 16–18 from the previous section.
2. Do not get any mud on the top of the cathode. If you do, carefully wipe it off, being careful not to grind it into the cathode.
8. Repeat the urine treatment following step 7 c.–g. with the second microbial fuel cell, but this time add 5 mL of urine to the soil. You might have to refill your transfer pipette several times. Make sure to spread the urine equally across the mud surface.
3. Starting the day after you add the urine, repeat steps 1–5 each day until it looks like the power output (the peak power) of both microbial fuel cells is stabilizing again (as described in step 6). Shortly after it stabilizes, the power output may then clearly change again.
1. Take these measurements at the same time every day.
2. For each day, for each MFC make a data table like Table 2 in your lab notebook to record your results and use them to determine the peak power. Note: It is possible that the resistor you use to determine the peak power will change slightly. Make a note of this in your lab notebook if it happens.
3. Depending on the exact conditions of your experiment—which can be affected by the soil you use—the power output could take anywhere from several days to over a week to stabilize after adding the urine.
1. When the power output has stabilized, the peak power may not change by more than about 10% for at least three days in a row. How much the power varies when it is "stabilized" can depend on the amount of power being produced by the MFC.
2. Overall, if it has been about two weeks after adding the urine and the peak power has not been steadily decreasing or increasing each day for at least the last week, then it has probably stabilized enough.
3. After it has stabilized, the power output may then clearly change again (steadily decreasing or increasing each day).
4. Tip: Making a graph of your data as you collect it for each MFC may help you see if the power output is stabilizing. If you do this, put the days on the x-axis and the power output (peak power) on the y-axis. Does the peak power appear to be stabilizing?
5. The time between LED blinks should also stabilize.

### Analyzing Your Results and Continuing Explorations

1. Make two graphs of your data for each microbial fuel cell, one showing how the power output changed over time and one showing how the frequency of LED blinks changed over time.
1. For the graph showing power output over time, put the number of days after setting up the MFC on the x-axis and the peak power output (in μW) on the y-axis.
2. For the graph showing the frequency of LED blinks over time, put the number of days after setting up the MFC on the x-axis and the blinks per second on the y-axis.
1. If you recorded the time between blinks in your data table, convert this to blinks per second by taking the data for the average seconds between blinks that you collected each day and calculate what 1 divided by this number is. For example, if your LED blinked an average of once every 15 seconds, 1 divided by 15 is 0.067, which is the number of blinks per second it made.
1. What happened to the power output and frequency of the LED blinks in both MFCs the day after adding urine? Was there an increase or decrease in power output? How large of a change was it?
2. How quickly did the measurements stabilize after adding urine? When they stabilized, were they higher or lower than they were originally, before adding the urine? Where did the power output stabilize in the microbial fuel cell with 1 mL urine compared to the other one with 5 mL urine?
3. When was the power output and blinking frequency the highest for each MFC? What was their peak power at this time?
4. Based on your results, do you think it would be feasible to use urine (and/or other human waste) to help generate power? Why or why not?
3. At this point in your experiment, there are several ways to continue your explorations. Here are a few starting points:
1. You could try repeating the entire experiment at least one more time. Is the change in power for each MFC similar each time?
2. You could try adding 1 mL and 5 mL of urine each time the MFC stabilizes (or right when the power output starts to change after it has stabilized). In both MFCs, do you see a change in power each time the urine is added? Is there a point at which additional urine does not change the power output?
3. You could repeat the entire experiment, but this time use even larger amounts of urine (such as 10 mL). Do you always see a positive effect on the power output for each initial amount of urine added or is there a urine volume that decreases the power output? If so, why?
4. Check out the Variations section for even more ideas for further exploration.

## Variations

Note: Carefully clean your microbial fuel cells before you start a new experiment. We recommend using tap water to rinse the electrodes while gently rubbing them (with gloves on) until the dirty water runs clear. Also completely rinse out the vessel with tap water. See the end of the Frequently Asked Questions for details.
• In this science project, you tested how the addition of urine affects the power output of an MFC. Can you think of other waste products that you could feed to the electrogenic bacteria? What about cow manure, food waste, or compost? Investigate what other waste products you can turn into electricity. Hint: You may want to look into what the soil bacteria (discussed in the Introduction) like to eat and the environments they thrive in.
• How does adding urine to the MFC—as you did in this science project—compare to adding nothing to the MFC and just letting it continue to run as you originally set it up? To try this out, you can use the second MFC as a control to run at the same time as the one that you add urine to. How does the power output of the MFC that has no urine added compare to the one that you added urine to?
• For additional ideas focusing on using human urine as a resource, or other waste products to create energy, see these related Science Buddies science project ideas:
• An unusual potential application for MFCs is using them to power medical devices that are implanted in a person. It is possible that the MFCs could run using sugar and oxygen from the person's blood. This is appealing because MFCs can run for a very long time, unlike batteries that would need to be replaced more often. You could investigate how well adding something similar to blood, such as blood meal (a plant fertilizer), affects the power output of the MFC. For more information on this type of application, see page 903 of this scientific paper:
• What other factors might affect and improve the power output of a microbial fuel cell? Investigate other parameters, such as temperature, soil moisture, or soil type, and the addition of other substances, like salt or sugar. Develop a well-reasoned hypothesis for what you think will happen when this factor is changed and create a way to test it. Which parameter do you think has the biggest effect on power output? If your MFC is generating between 80 µW and 199 µW, it is doing really well, and if it is generating 200 µW or more, it is doing amazingly well! Examples for investigating soil conductivity, soil type, and the addition of sugar as substrate are given in the Science Buddies science projects Spice Up the Power of a Microbial Fuel Cell with a Dash of Salt, Turn Mud into Energy With a Microbial Fuel Cell, and How Do Bacteria Produce Power in a Microbial Fuel Cell?.

## Share your story with Science Buddies!

One easy way to figure out how to troubleshoot your experiment is reviewing the flow chart in Figure 9. You will find more detailed information in the FAQ section below.

Figure 9. Flow chart for troubleshooting the microbial fuel cell experiments.
Q: The LED on the microbial fuel cell has never blinked. What should I do?
A: Even if the LED is not blinking, you should still take and analyze your peak power output measurements and you may be able to continue with the experiment, as described in the Measuring Power Output and Adding Urine section in the Procedure. If your peak power output stabilizes around at least 4 μW, then this is enough power for you to continue with the experiment. It is possible that the microbial fuel cell is making enough power for you to continue with the experiment even though the LED is not blinking. There are several reasons why the LED might not be blinking:
• If the hacker board is not set up correctly, the LED will not blink. You should confirm that everything is set up as described in the Setting Up the Microbial Fuel Cells section of the Procedure.
• The LED might not have enough power to blink. You will need a peak power output of at least 4 μW before the LED starts to blink, and the initial blinking may only be about once every 30 seconds, so you will need to closely watch the LED to see it blink.
• Depending on the type of topsoil you are using, it may take about 3–7 days for the LED to start blinking, if everything is properly in place. Some types of topsoil might make the microbial fuel cell take longer to blink than other types of topsoil because it may take longer for enough power to be made to light up the LED. Make sure that you are not using any topsoil with little white foam balls, vermiculite pieces, or perlite, since these can aerate the soil and inhibit the desired bacteria from growing (they do not want to be exposed to oxygen). Also avoid types of topsoil with peat moss or topsoil that has become completely dry.
• The temperature of the room that the microbial fuel cell is in can significantly affect how well the bacteria grow, which affects whether (and how quickly) the LED blinks. The microbial fuel cell should be kept indoors, at normal room temperatures (about 19–25° Celsius [C], or 66–77° Fahrenheit [F]), in the same location the entire time after you set it up. If the room gets too cold, the bacteria may not grow well. If the microbial fuel cell is moved to a different location, this could disrupt the growth of the bacteria.
• Even if the hacker board is set up correctly, some of the wires might be loose or may not be making good electrical contacts in the hacker board. You can try taking the wires out of the microbial fuel cell and then putting them back into the correct positions, and/or gently jiggling the wires around in the slots in the hacker board. Also make sure that none of the exposed parts of the wires are touching each other.
• It is possible that the LED has become damaged. You can check to make sure the LED is working by hooking it up to a battery, as shown in Figure 5 of the science project idea: See the Light by Making a Cell Phone Spectrophotometer.
Q: The peak power output of the microbial fuel cell seems low. What might be the problem and what can I do?
A: If your peak power output stabilizes around at least 4 μW, then this is enough power for you to continue with the experiment, as described in the Measuring Power Output and Adding Urine section in the Procedure. It may take about 7–14 days for the peak power output to reach at least 4 μW, depending on the topsoil and other conditions used. If your peak power output does not reach at least 4 μW by 7–14 days after setting up the microbial fuel cell, there are several possible reasons for this:
• If the hacker board is not set up correctly, it will not produce power. You should confirm that everything is set up as described in the Setting Up the Microbial Fuel Cells section of the Procedure.
• Even if the hacker board is set up correctly, some of the wires might be loose or may not be making good electrical contacts in the hacker board. You can try taking the wires out of the microbial fuel cell and then putting them back into the correct positions. Also make sure that none of the exposed parts of the wires are touching each other.
• If it looks like the peak power output is still increasing each day, it may just take a few more days for the microbial fuel cell to reach 4 μW.
• The soil you are using might not work well in the microbial fuel cell. First be sure that you are using topsoil, and not potting soil. Make sure that you are not using any topsoil with little white styrofoam balls, vermiculite pieces, or perlite, since these can aerate the soil and inhibit the desired bacteria from growing (they do not want to be exposed to oxygen). Also avoid types of topsoil with peat moss or topsoil that has become completely dry.
• If there are air bubbles trapped in the damp topsoil in the microbial fuel cell, this can prevent the bacteria from growing well because they do not want to be exposed to oxygen. When packing the mud in the microbial fuel cell, pat down the mud and electrodes, as described in the Setting Up the Microbial Fuel Cells section of the Procedure, so that you do not have any trapped air bubbles in the mud. Air bubbles may also be caused by small, hard particles (such as rocks, pebbles, vermiculite, twigs, etcetera) being trapped in the mud, so anything like this should be removed from the topsoil before adding it to the microbial fuel cell.
• Check if your soil in the microbial fuel cell is still moist. The soil needs to be moist for good electron conductivity and for the bacteria to grow. If your soil is dry, carefully open the microbial fuel cell and mix distilled water into the soil until it is moist again. However, make sure not to disturb the anodic biofilm.
• If the temperature of the room that the microbial fuel cell is in is very cold, the bacteria may not grow well, which would decrease the peak power output. The microbial fuel cell should be kept indoors, at normal room temperatures (about 19–25° C, or 66–77° F), in the same location the entire time after you set it up. Also, if the microbial fuel cell is moved to a different location (particularly if it is at a different temperature), this could disrupt the growth of the bacteria.

Even if the microbial fuel cell is not making more than 4 μW, this may still be an interesting result, especially if you are testing conditions different from the ones described in the main project idea. Keep in mind that a science project does not have to work as anticipated in order to be real, good science. It is important to communicate (on your Science Fair Project Display Boards, report, or however you are presenting your project) your question, your hypothesis, what you anticipated would happen, then what you actually saw (such as your peak power output measurements over time), your questions about what was happening, and your attempts to troubleshoot. You could also communicate any possibilities of what could be happening and how you would test that (if you do not have enough time to test it now).

Q: Is the voltage made by my microbial fuel cell too low?
A: When you take the voltage measurements with the different resistors each day, the voltage measurements need to be converted into power (in microwatts, or μW). This is described in the Procedure in the section Measuring Power Output and Adding Urine. The power output depends on the resistors you use, so you cannot determine how well the microbial fuel cell is performing by just looking at the voltage measurements alone; they need to be converted into power for them to be meaningful. Once you have converted the voltage measurements into power outputs and determined your peak power output, you can determine whether the peak power output seems low or not. If your peak power output stabilizes around at least 4 μW, then this is enough power for you to continue with the experiment, as described in the Measuring Power Output and Adding Urine section in the Procedure. It may take about 7–14 days for the peak power output to reach at least 4 μW.
Q: The LED on the microbial fuel cell was blinking, but now it has stopped and this is unexpected. Why did this happen?
A: Even if the LED stops blinking, you should still continue to take and analyze your peak power output measurements and you may be able to still do the experiment, as described in the Measuring Power Output and Adding Urine section in the Procedure. If the LED was blinking one day but then stopped blinking the next, there are several possible reasons for this:
• If the hacker board is not set up correctly, the LED will not blink. You should confirm that everything is still set up correctly, as described in the Setting Up the Microbial Fuel Cells section of the Procedure.
• Even if the hacker board is set up correctly, some of the wires might be loose or may not be making good electrical contacts in the hacker board. You can try taking the wires out of the microbial fuel cell and then putting them back into the correct positions, and/or gently jiggling the wires around in the slots in the hacker board. Also make sure that none of the exposed parts of the wires are touching each other.
• The LED may no longer have enough power to blink. You will need a peak power output of at least 4 μW, for the LED to blink, and the blinking may be as infrequent as about once every 30 seconds, so you will need to closely watch the LED to see it blink.
• If the temperature of the room that the microbial fuel cell is in is very cold, the bacteria may not grow well, which would decrease the peak power output and affect whether the LED blinks. The microbial fuel cell should be kept indoors, at normal room temperatures (about 19–25° C, or 66–77° F), in the same location the entire time after you set it up. Also, if the microbial fuel cell is moved to a different location (particularly if it is at a different temperature), this could disrupt the growth of the bacteria.
• It is possible that the LED has become damaged. You can check to make sure the LED is working by hooking it up to a battery, as shown in Figure 5 of this science project idea: See the Light by Making a Cell Phone Spectrophotometer.
• Check if your soil in the microbial fuel cell is still moist. The soil needs to be moist for good electron conductivity and for the bacteria to grow. If your soil is dry, carefully open the microbial fuel cell and mix distilled water into the soil until it is moist again. However, make sure not to disturb the anodic biofilm.

Keep in mind that a science project does not have to work as anticipated in order to be real, good science. You should always record your observations, even unexpected ones, and think of ways to explain why they happened.

Q: The peak power output does not seem to be stabilizing. Should I be concerned?
A: About 7–14 days after setting up the microbial fuel cell, the peak power output should stabilize to around at least 4 μW. For the peak power output to be considered "stabilized," it should not change by more than about 10% for at least three days in a row. But even if your peak power output changes by a little more than this, do not worry; if it has been at least 14 days and the peak power looks like it is stabilizing when you graph it each day (i.e., it is not steadily increasing or steadily decreasing from day to day), then it has probably stabilized enough. Keeping this in mind, if it still does not look like the peak power output is stabilizing, this could be because some conditions have changed (i.e., the temperature has changed or the fuel cell has been moved to a different location). It is very important that conditions are kept as consistent as possible for the peak power output to stabilize and to do the rest of the experiment using the fuel cell, as minor changes in conditions can significantly affect the power output.

If the power output does not seem to be stabilizing after the addition of urine, try waiting a little longer. It could take about two weeks for the power output to stabilize after adding urine. If it has been about two weeks after adding urine and the peak power is not steadily decreasing or increasing each day, then it has probably stabilized enough.

Q: After I set up the microbial fuel cell, the peak power output was increasing, and then decreased before I added any urine. Should I be concerned?
A: Within the first 7–14 days of setting up the microbial fuel cell, it is normal to see some fluctuations in the peak power output. This may include seeing the peak power output increasing, and then decreasing a little. After about 7–14 days, the peak power output should appear to stabilize. If this does not appear to happen, refer to the other relevant questions in this FAQ.
Q: What is the purpose of using the different resistors?
A: You need to use multiple resistors so that you can determine what the peak power output of your microbial fuel cell is. The power output depends on which resistors you use, and you need to determine which resistor gives you your peak power output, as described in the Measuring Power Output and Adding Urine section of the Procedure. The resistor that gives the peak power output is closest to the internal resistance of the microbial fuel cell, and this depends on the type of topsoil you are using and other factors.
Q: Why do I need to wait until the peak power output has stabilized before adding the urine?
A: If you do not wait for the peak power output to stabilize before adding urine then you will not know how the addition of urine changed the peak power output because it would have still been fluctuating. You would not be able to know for certain whether any changes in peak power output you see, after adding the urine, would then be due to normal fluctuations of the microbial fuel cell or actually to the addition of the urine.
Q: Why is urine added to the microbial fuel cell?
A: As discussed in the Background, urine contains nutrients, and when the urine is added to the soil, these nutrients should be transferred to the bacteria in the soil. The bacteria should consume the nutrients and grow. Growing bacteria should produce more electricity in the MFC. This means that initially when urine is added to the MFC, there should be an increase in the peak power output, but eventually if too much urine is added, it may kill the bacteria and decrease the peak power output.
Q: During the voltage sweep the multimeter readings fluctuate a lot and do not seem to stabilize. Should I be concerned?
A: It is important to leave the resistor plugged in at least 5 minutes before you take the voltage measurements. It is normal to see the voltage readings decreasing slightly over the period of a few seconds. In this case watch the readings on the multimeter for a few seconds more until they stabilize (and stay the same for a few seconds). Use the stabilized value. If the voltage readings fluctuate randomly over a large voltage range, check your wire connections. If your microbial fuel cells already run for a long time (longer than 4 weeks), there might also be the chance that the internal connections inside the hacker board got worn out and the hacker board is damaged. In this case you need to replace the hacker board or end your experiment.
Q: Is there a way to clean out the microbial fuel cell without damaging it so that I can use it in another experiment?
A: Yes, the microbial fuel cell can be cleaned out without damaging it. To do this, carefully rinse the vessel and electrodes with tap water. The electrodes should be gently rubbed while rinsing them until the water running off of them is not dirty anymore. Also, to make sure you are starting with a clean slate of bacteria for each test, the vessel and electrodes should be quickly rinsed with 70% isopropyl rubbing alcohol, which should kill the bacteria. To do this, you can put the electrodes in the vessel (using it as a cup) and fill it with just enough alcohol to submerge the electrodes. Get some of the rubbing alcohol on the inside sides of the vessel while you do this, too. Then pour off the rubbing alcohol and briefly rinse the electrodes and vessel with tap water (so the rubbing alcohol does not remain on these parts). Make sure to follow all proper safety precautions when using the rubbing alcohol.

The Ask an Expert Forum is intended to be a place where students can go to find answers to science questions that they have been unable to find using other resources. If you have specific questions about your science fair project or science fair, our team of volunteer scientists can help. Our Experts won't do the work for you, but they will make suggestions, offer guidance, and help you troubleshoot.

If you have purchased a kit for this project from Science Buddies, we are pleased to answer any question not addressed by the FAQ above.

1. What is your Science Buddies kit order number?
2. Please describe how you need help as thoroughly as possible:

Examples

Good Question I'm trying to do Experimental Procedure step #5, "Scrape the insulation from the wire. . ." How do I know when I've scraped enough?
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Bad Question I don't understand the instructions. Help!
Good Question I am purchasing my materials. Can I substitute a 1N34 diode for the 1N25 diode called for in the material list?
Bad Question Can I use a different part?

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