For decades, water pollution has caused major crises worldwide. A significant amount of water contamination is caused by illegally deposited chemicals and sewage in rivers and lakes. This is an investigation of the efficiency of a constructed wetland filtration system in the removal of E. coli bacteria from water, since deadly cultures of this bacteria are often found in sewage. Three models were built: one, containing only Giant Bur-reed, another of Arrowhead, and a third without any plants to compare the plants' rates of absorption with.

Hypothesis:

If the contaminated water is put through the artificial wetland system, then much of the E. coli will be absorbed through the roots of the plants. Taking the large roots visible from the Giant Bur-reed into account, it will absorb bacteria at a greater rate than the Arrowhead.

Materials:

• 12 dormant Arrowhead plants
• 12 dormant Giant Bur-reed plants
• 3 plastic containers (34cm x25 cm x15cm)
• 10L of bottled spring water
• 9mL of Luria Broth inoculated with K12 E. coli
• 45 petri dishes
• 3 water pumps
• three 20 cm plastic hose pieces
• 12L of sterilized soil
• crock pot
• 3 grow lights
• small jar for preliminary test
• microscope
• 00mL MacConkey's Agar
• automatic balance and weighing dish
• autoclave
• large flask
• 30 test tubes
• 30 tips
• 90mL Luria Broth
• 1 large pipette and several Pasteur pipettes
• 2 digital pipettes
• Bunsen Burner
• several spreaders
• bleach and water solution
• permanent marker

Procedure:

I) Soil Steriliztion (Cole 470)

1. Bring 500mL of distilled water to a boil in a large stock pot.

2. When it boils, add 6L of dry soil. Cover tightly. Return to a boil.

3. Turn heat down to prevent boiling over. After five minutes, turn off the heat.

4. Repeat process once, to sterilize a total of 12L of soil.

1. Place 4L of sterilized soil into each of the containers.

2. Place12 plants of one species in each container, about two inches beneath the soil, so that the roots/bulbs are completely covered. Leave one container to use for a control later in the experiment.

3. Add 3L of distilled water into each container.

4. Install and turn on pumps.

5. Allow three weeks for plants to establish themselves beneath the grow lights. Check water level daily and replace amount lost by evaporation.

1. Measure out 25g of MacConkey's Agar using the automatic balance. Mix with 500mL of water and place in the autoclave for sterilization.

2. Using the pipette, measure out 3mL of Luria broth per test tube for thirty tubes. Place in the autoclave for sterilization.

3. Place sterilized and cooled Agar into petri dishes. Remove bubbles using Bunsen burner, and cover.

4. Using Pasteur pipettes, take samples from each of the model wetlands into sterilized test tubes, and cover.

5. Add 3mL of bacteria to each container. Obtain samples at the following intervals:

   0 minutes
   24 hours
   48 hours

1. Add a drop of Luria broth to each of the Petri dishes, discarding test tubes in between separate samples.

2. If necessary for receiving countable results, create a 1:100 L dilution of a selected sample and Luria broth in a sterilized test tube, using digital Pasteur pipettes.

3. Measure desired sample volume using digital Pipette, and release onto petri dish. Sterilize test tube in between uses, by passing through Bunsen burner flame.

4. Using sterilized spreader, spread dilutions over hardened Agar, and cover.

5. Repeat process for each sample taken. Change volumes placed in Petri dishes when needed to attain a countable number of colonies.

6. After plating samples, stack and leave for one day. Divide Petri dishes into quarters using a permanent marker and count colonies. Record results.

   Independant Variables

   • the amount of bacteria placed in each of the containers
   • the intervals at which samples are taken
   • the amount of light received by each container
   • the number of dishes plated per sample

   Dependant Variables

   • the amount of bacteria absorbed between intervals
   • the total amount of bacteria absorbed

   Observations and Analysis

   A: Giant Bur-reed

   B: Arrowhead

   C: Container without plants

   Formulas: Undiluted Sample: cells/mL= (#colonies)(1mL/ volume plated) Diluted Sample: cells/mL= (#colonies)(dilution factor)(1mL/ volume plated) *The first sample after the inoculation were plated at 10 L, since an overnight culture of E. coli contains about 109 cells/ml, and an amount was added to the containers that would give us a countable number in 10ul. The number of colonies visible previous samples were used to predict the volume that would need to be plated to obtain a countable number in the next sample. For example, Model A showed a decrease of roughly 5/6 within the first 24 hours. It was predicted that the plants would continue to absorb around this amount each 24 hours, so a similar volume was chosen for subsequent samples.