Abstract

Objective

The goal of this experiment is to compare the effects of added nitrogen fertilizer vs. nitrogen-fixing bacteria on the growth of clover.

Introduction

Plants require more than just water and sunlight to grow. They also require many nutrients found in the soil. One of the most important nutrients required for plant growth is nitrogen. Nitrogen is used to build plant proteins and nucleic acids, including DNA.

Nitrogen is found naturally in the atmosphere. In fact, it makes up about 78% of the atmosphere! But this form of nitrogen (N₂) cannot be used by plants. Nitrogen can be combined chemically with oxygen or hydrogen to form types of nitrogen compounds that plants can use. These nitrogen compounds can be added to the soil in the form of ammonium (NH₄⁺) and nitrate (NO₃⁺) fertilizer. Plants grow well when fertilizer containing nitrogen is added to the soil, but this method can be expensive and has to be repeated each time the nitrogen in the soil is used up.

Bacteria to the rescue! Bacteria are small, single-celled organisms that live in nearly every environment on Earth. From the freezing cold of Antarctica to the boiling heat of hot springs in Yellowstone National Park, some of these organisms are able to live in extreme environments and have many amazing capabilities. Some species of bacteria are able to turn milk into cheese while others can reproduce in less than twenty-four hours. Rhizobia, the type of bacteria that you will study in this experiment, can turn the nitrogen in the soil into usable nitrogen compounds like ammonium and nitrate ions. This is called nitrogen-fixation. These bacteria can attach themselves to the roots of some plants, forming little growths called nodules. The rhizobia receive nutrients and protection from the plant roots and the plants get their fill of nitrogen. This type of mutually beneficial relationship is called a symbiotic relationship. Legumes, and clover in particular, readily form this symbiotic relationship with rhizobia.

In this experiment, you will grow clover plants in soil with no nitrogen added, in soil with nitrogen fertilizer added, and in soil containing nitrogen-fixing bacteria (in this case, a species of rhizobia called Rhizobium legominosarium, or R. legominosarium). You will monitor the nitrogen levels in each type of soil using a nitrogen testing kit. You will observe the effects of nitrogen on the health of the clover plants by measuring the increase in biomass of each plant during the experiment.

Terms, Concepts, and Questions to Start Background Research

• Nutrients
• Nitrogen
• Compounds
• Bacteria
• Rhizobia
• Nitrogen-fixation
• Nodules
• Symbiotic relationship
• Legumes
• Biomass

Questions

• What nutrients do plants need to survive?
• How do nitrogen-fixing bacteria change nitrogen into ammonium and nitrate ions?
• What is symbiosis and what types of organisms form symbiotic relationships?
• What types of plants form symbiotic relationships with nitrogen-fixing bacteria?
• How do root nodules form?

Bibliography

These websites have more information about nitrogen-fixing bacteria:

• Wojciechowski, Martin F. and Mahn, J. (2006). Diversity in Legume Root Nodules. Retrieved May 10, 2010, from http://tolweb.org/notes/?note_id=3948
• Microbe Zoo. (n.d.). The Root Cellar. Retrieved May 10, 2010, from http://microbezoo.commtechlab.msu.edu/zoo/zdrmain.html

This site provides chemical information on the nitrogen cycle:

Garden Centre Direct, Kybotech LTD. (2010). The Nitrogen Cycle Explained. Retrieved May 10, 2010, from http://www.gardencentredirect.co.uk/Article/the-nitrogen-cycle-explained

This website provides information about symbiosis and nitrogen fixation.

• Kimball, J. (2010, March 12). Symbiotic Nitrogen Fixation. Retrieved May 10, 2010, from http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/N/NitrogenFixation.html

Materials and Equipment

Caution: Adult supervision is required during use of the nitrogen fertilizer.

• Small pots (at least 20); make sure the pots have holes in the bottoms to allow the plants to "breathe."
• Watering can
• Potting soil, make sure to chose one with a low amount of nitrogen compounds. If you're unsure what to chose, ask a specialist at a local nursery or garden store for help. Make sure to explain your experiment to them.
• Masking tape
• Permanent marker
• Clover seeds (1 packet); can be purchased from a garden store, or ordered online from Carolina Biological Supply Company item # 154720B.
• Rhizobium innoculum (1 packet); available online from Carolina Biological Supply Company item # 154719B.
• Small re-sealable plastic bags (2); snack size.
• Measuring spoon; ¼ teaspoon size.
• Paper towel
• Nitrogen fertilizer
• Grow light (optional)
• Watering can
• LaMotte N-P-K Soil Test Kit; available online from Carolina Biological Supply Company item # 181875.
• Scale, metric, such as the Fast Weigh MS-500-BLK Digital Pocket Scale, 500 by 0.1 G, available from Amazon.com
• Beaker
• Lab notebook

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Experimental Procedure

Note: Adult supervision required during use of nitrogen fertilizer.

Innoculating the Clover Seeds with Rhizobia

1. Open the package of clover seeds and pour half of the package into each re-sealable plastic bag.
2. Using the measuring spoon, add ¼ teaspoon of water to each plastic bag. The water will help the rhizobium innoculum to stick to the clover seeds. Although you won't be adding rhizobia to both batches of seeds, you want to treat the seeds as similarly as possible, that is why you add water to both batches of seeds. Using masking tape and permanent marker, label one of the plastic bags: rhizobia coated.
3. Pour approximately 1/4 of the rhizobium innoculum into the plastic bag labeled rhizobia coated.
4. Seal both plastic bags and shake to thoroughly mix the water, clover seeds, and (in one of the two bags) the rhizobium innoculum.
5. Spread out two paper towels on a flat, dry surface. Pour each plastic bag of seeds on to its own paper towel. Label the paper towel with the rhizobia coated seeds.
6. Wait 30 minutes for the seeds to dry before proceeding to the planting part of the procedure.

Planting and Collecting Data

1. Put equal amounts of soil in each pot.
2. Moisten the soil in each pot with equal amounts of water. You want the soil to be moist, but not soupy.
3. Using the masking tape and permanent marker, label the pots:
1. no nitrogen added; at least 5 pots
2. nitrogen fertilizer; at least 5 pots
3. rhizobia; at least 5 pots
4. rhizobia + nitrogen fertilizer; at least 5 pots
4. Plant seeds in each pot according to seed packet instructions.
1. In the pots labeled "no nitrogen added" and "nitrogen fertilizer" plant untreated clover seeds.
2. In the pots labeled "rhizobia" and "rhizobia + nitrogen fertilizer" plant the rhizobia coated clover seeds.
5. Place plants near a sunny window or under a grow light.
6. Set a schedule for watering. Soil should be kept moist.
7. Water the "no nitrogen added" and "nitrogen-fixing bacteria" pots with regular water. Water the "nitrogen fertilizer" pots with water and nitrogen fertilizer at the brand of fertilizer's recommended concentration. You may not need to add fertilizer at each watering.
8. Clover will grow to maturity in 5-6 weeks. When clover plants are mature, proceed with experiment.
9. Use the soil test kit to measure the amount of nitrogen in the soil of each pot. Remember to record your data in your lab notebook.
10. After these measurements are complete, carefully remove each plant from the soil and shake off any excess soil from the roots.
11. Measure the total biomass of each plant and record data in your lab notebook. For more information on scientifically measuring plant growth see the Science Buddies How-to page, Measuring Plant Growth.
12. Graph the average nitrogen levels in each category (remember, there are a total of 4 categories as listed in step 3) and the average biomass of the clover grown in each category. Which category had the highest levels of nitrogen? Which category produced the greatest biomass of clover? Was there any noticeable difference in the health or appearance of the clover grown with or without nitrogen fertilizer? Did inoculating the clover with rhizobia affect either the nitrogen levels in the soil or the total biomass?

Variations

• Compare different amounts of nitrogen fertilizer to the clover inoculated with the nitrogen-fixing bacteria.
• Test the abilities of other plants to form a symbiotic relationship with nitrogen-fixing bacteria.
• Test different species of nitrogen-fixing bacteria to see which type produces the most nitrogen in the soil or the healthiest plants.

• For more science project ideas in this area of science, see Plant Biology Project Ideas.

Credits

La Né Powers

Edited by Sandra Slutz, PhD, Science Buddies

Last edit date: 2011-11-18 12:00:00