Key concepts
Microorganisms
Soil
Biology
Oxygen
Sunlight
Ecosystem

Introduction
Our planet recycles and reuses everything on it needed to support life. It's an amazing, giant recycling system called the biogeochemical cycle. You can actually model this on a small scale by using a plastic bottle and mud to build what's called a Winogradsky column. In this activity you'll build your own Winogradsky columns and investigate how including different nutrients affects which soil microorganisms flourish and which fail.

Background
To live and grow, organisms need specific nutrients nearby for them to eat—just like we do. Most of these nutrients are continuously being moved through a biogeochemical cycle, which transports nutrients and other chemicals through the living and nonliving parts of Earth—for example, iron that was once in a rock might later enter the soil where it can be absorbed by a plant and then eaten by an animal. The biogeochemical cycle is a closed system, which means that the nutrients aren't lost or created; they're continuously reused and recycled.

A given ecosystem might have several biogeochemical cycles going on within it. Some important nutrients that are recycled through an ecosystem are oxygen, carbon and sulfur. Different soil microbes play key roles in recycling these and other nutrients. A tool that microbiologist Sergei Winogradsky invented to study these processes was a long, sealed column of muddy soil, now called a Winogradsky column. Within one of these, different gradients form. For example, over time there is more oxygen at the top of the column than at the bottom. These gradients affect where different microbes can live within the column.

Materials
• Four clean plastic water bottles, 500 milliliters each. Bottles that have smooth sides (not ridges), and are taller and narrower in shape should work best.
• Scissors
• Knife (optional)
• Permanent marker
• Ruler
• Rubber boots and old clothes that can get muddy (optional)
• Access to a muddy stream, pond, lake or marsh
• Gloves
• Shovel or trowel
• Two buckets
• Newspaper or plain paper (shredded)
• An egg
• Two bowls
• Two large mixing bowls
• Measuring cup
• Wide stick, for packing mud into the bottles
• Measuring teaspoon
• Adjustable desk lamp with 13-watt compact fluorescent bulb (optional)
• An empty surface, such as on a desk or table, that is at warm room temperature (about 72 to 78 degrees Fahrenheit). It should not receive any direct sunlight but if you're not using a desk lamp, the area should be near a very sunny window.
• Plastic wrap
• Plastic trash bags or grocery bags
• Four rubber bands
• Cardboard box or brown paper bag
• Flashlight

Preparation
• Remove any wrappers from your four water bottles as best you can.
• Carefully cut the tops off your bottles. You may need to use scissors and a knife to do this, and an adult might need to help. Cut the top off right where the bottle starts curving inward near the top. Save the cut-off tops to use as funnels later.
• Using a permanent marker, make a small mark where your bottle would be about 85 percent full. For example, if your bottle is six inches tall, you would make a mark at about five inches up from the bottom. You will fill the bottles to this level with mud.
• Label each bottle with what will be added to its mud. One will have newspaper added, one will have egg yolk added, and two will only hold plain mud (with nothing added). One of the plain bottles will be kept in the dark, the other three bottles will be exposed to light.
• Go collect some mud from a muddy stream, pond, lake or marsh. Use caution around the water and always have adult supervision. (You may want to first put on some rubber boots and old clothes that can get muddy.) Put on gloves and fill one bucket with about one-half to one gallon of mud, scooping it mostly from just below the water's surface.
• In the other bucket, collect some water (one-half gallon at most) from the same location.
• Bring your buckets home and go wash up if needed! (Until you use them, keep your buckets shaded so they don't get too hot.)

Procedure
• In a bowl cut about a quarter sheet of newspaper or plain paper into thin strips, and then cut the strips into tiny rectangles. These will be a source of carbon for the microbes in the mud. How do you think adding carbon will affect the microbes that grow in the Winogradsky column?
• In a second bowl add an egg yolk (raw or hard-boiled). If it is hard-boiled, crush the yolk into a  mash. If it is raw, be sure to wash your hands after handling it, as raw eggs may contain salmonella bacteria. The yolk will be a source of sulfur for the microbes in the mud. How do you think adding sulfur will affect the microbes that grow in a Winogradsky column?
• Put on some gloves and get your bucket with your mud. While stirring, slowly add the water you collected to the soil until your mixture becomes like a milk shake. Also while stirring, be sure to pick out all sticks, leaves and rocks from the mud.
• In a large mixing bowl blend a little more than two cups of the mud and about one third of the egg yolk. Using a cut-off bottle top as a funnel, pour about one inch of the mud mixed with egg yolk into the bottle labeled as having egg yolk added. Tap the bottle on a hard surface to pack the mud down and use a wide stick to pack the mud more. Continue adding about one inch of mud at a time and packing it down until you are at the 85 percent full mark you made. Set the bottle aside.
• In another large mixing bowl blend a little more than two cups of the mud from the bucket (remix if water has settled out) and one teaspoon of the shredded newspaper or paper. Using a cut-off bottle top as a funnel, pour and pack about one inch of the mud at a time into the correctly labeled bottle until you are at the 85 percent mark. Set the bottle aside.
• Take the two remaining bottles, which you should have labeled as having plain mud. Into the two bottles funnel and pack in mud directly from the bucket of mud (remix if water has settled out) until they're 85 percent full.
• After sitting about 30 minutes the water should be about 0.2 to 0.8 inch deep on top of the mud in each bottle. Carefully add more water or remove some, as needed. Leave at least 0.2 inch of empty space at the top.
• Find an empty flat surface, such as a desk- or tabletop, that is at warm room temperature (about 72 to 78 degrees F). It should not receive any direct sunlight. If you're not using a desk lamp, the area should be near a very sunny window. Cover the surface with plastic trash or grocery bags to protect it.
• Carefully move your bottles to the surface, being careful not to spill them! Cover each bottle with plastic wrap, secured with rubber bands. Your Winogradsky columns are now ready for testing!
• If you're not using a desk lamp, arrange the three Winogradsky columns (that will receive light) so that they'll receive a lot of light, but they should not be exposed to direct light. If you're using a lamp, arrange the three columns so that they're 20 inches away from the lightbulb. For those bottles that are being lit by lamp, write a small "L" on the sides of the bottles that are facing the lamp. What do you think will happen on the sides facing the light?
• Set the Winogradsky column with plain mud that won't receive light on a surface at room temperature. Then either place a cardboard box upside down over the column, or put it in a brown paper bag, so that no light reaches the bottle. What do you think will happen in the column that doesn't receive light?
• For the next six to eight weeks leave the Winogradsky columns where you set them. If you're using one, keep the desk lamp on 24 hours a day. (If a plastic wrap lid comes loose, just reattach it with a rubber band.) Observe the columns once a week, looking for color changes in them. Each area of coloring should be a group of the same type of microbes. When observing the columns, try turning off the lights and shining a bright flashlight at the columns—this can help you see the colors better. What colors do you see appear in the columns? Where in the columns do they appear? How do the columns look different from one another? Do you see any worms, shrimp, snails or other larger organisms in the columns? How do the columns change over time? What do you think your results have to do with what was added (or not added) to the columns?
• Tip: You should see some green coloring appear on some columns after one to two weeks. You may need to look closely for it. If you do not see any green coloring, the columns may not be receiving enough light. You could try moving them closer to the light source.
• Extra: Do this activity again, but this time test multiple bottles for each condition. For example, test three Winogradsky columns with egg yolk. How reproducible are your results? Is there a lot of variation between the different columns that were set up the same way?
• Extra: You could try testing several different sources of mud or soil to see if the microbial growth will be different from location to location. You could even try some beach sand. What do you think your results tell you about the soil quality and microbes that live at each site you test?
• Extra: Test some different kinds of additives to look for microbes that live in unique and challenging environments. For example, you could test increasing amounts of salt in a series of Winogradsky columns to test for salt lovers (called halophiles) or place columns at different temperatures to find microbes that like heat (near a heat vent) or cold (in the refrigerator). Can you select for microbes that live in more extreme conditions?

Observations and results
Did the columns that were in the light make areas of green coloring on the sides facing the light whereas the column in the dark remained dark brown? Did the three columns that were in the light create color patterns that were somewhat different from one another?

Over time gradients of different nutrients should have formed in the Winogradsky columns. These gradients affect where different microbes grow within the columns. For example, over time there's more oxygen at the top of a column that at the bottom, and this means that microbes that can tolerate or make oxygen will be at the top. Microbes that cannot tolerate free oxygen (called anaerobic bacteria) will be further down. Similarly, microbes that need light to make energy (via photosynthesis or a similar process) will need to live where they can get light in the column.

After about one to two weeks, depending on how much light the columns receive, some green coloring should appear in the columns receiving light on the illuminated sides. This is mostly due to cyanobacteria and algae, which need light. The column in the dark should remain dark brown. In the column that had egg yolk you may have seen areas of darker green, purple, and/or black coloring develop over time near the bottom—these colorings could be groups of certain anaerobic bacteria: green sulfur bacteria, purple sulfur bacteria and sulfate-reducing bacteria, respectively. Sulfate-reducing bacteria actually eat sulfur and make hydrogen sulfide gas, which is eaten by the green and purple sulfur bacteria. In the column that had newspaper you may have seen some areas of brown, orange, red or purple near the middle—these colorings could be groups of purple nonsulfur bacteria, which need a carbon source to thrive. You may have seen worms, snails, shrimp or other small organisms in the water, but probably not many (if any) in the bottle with the egg yolk, because hydrogen sulfide is toxic to most organisms!

Cleanup
Be sure to wash your hands and anything else that came into contact with the raw egg, because they can carry salmonella. Also wash your hands after handling the mud. When you are done with your Winogradsky columns, with permission you can dump the mud outside (such as in a composter or back in a muddy area).

More to explore
Building a Winogradsky Column Video Demonstration, from NASA Quest
The Winogradsky Column, from Microbial Life
Winogradsky Columns, from The Pennsylvania State University
Growing a Soil Menagerie, from Science Buddies

This activity brought to you in partnership with Science Buddies