This project contains:
           Abstract (a summary of the project)
           Background Information
           Problem
           Hypothesis
           Procedure
           Control
           Constants
           Independent Variable
           Dependent Variable
           Data
           Pictures
           Analysis/Discussion
           Conclusions
           10 Things That Were Learned
           References Sited

  Abstract/Summary  

         For our project we studied the effect of Rhizobium Leguminosarum on the growth of pea plants. Using an inoculum, we introduced the bacteria to one set of field pea seeds. The other set was left without bacteria. We then planted the seeds and let them grow. For a month we observed our plants' growth and measured their height in centimeters. The data that we gathered was consistent with our hypothesis. The plants that had been inoculated with the Rhizobium grew faster, taller, and looked more vibrant and healthy than the plants without bacteria. We also discovered nodules on the roots of the inoculated plants. These observations are clearly the result of the bacteria. As our research paper explains, Rhizobium Leguminosarum is a nitrogen-fixing bacteria. Bacteria of the Rhizobia classification stimulate the plant to form nodules on its roots. Once the bacteria enter these nodules, they convert atmospheric nitrogen to ammonia, a nitrogen compound that plants are able to use. The plant then uses ammonia to assemble amino acids, the building blocks of proteins. As a result, plants that receive excess nitrogen will grow faster, higher, and healthier than those plants that do not.

  Background Information  

         This project is a study of a symbiotic relationship between a field pea plant (leguminous) and a nitrogen-fixing bacterium, Rhizobium Leguminousarum. A symbiotic relationship is an association or a relationship where both organisms mutually benefit. Once a symbiotic relationship is established between two or more organisms, the organisms often become incapable of living without their symbiotic partner(s). Rhizobium Leguminosarum is a strain of bacteria that converts atmospheric nitrogen to ammonia. Ammonia is used by the plant to make amino acids - these amino acids are then assembled to make proteins. Some of the carbohydrates manufactured by the plant during photosynthesis are transported to the nodules where they are used as a source of food by the Rhizobium. The Rhizobium also uses some of the carbohydrates as a source of hydrogen in the conversion of atmospheric nitrogen (N2) to ammonia (NH4+). (Soil improvement with legumes, 5A) The Rhizobium combine this hydrogen with the atmospheric nitrogen in the conversion process.

         Bacteria of the Rhizobium strain are present in most soils. It is one of the most beneficial microbes living in the soil. There is a certain process that must be followed in order to successfully introduce Rhizobium to a legume.

1. The Rhizobium bacteria must encounter the right legume (Rhizobium can only infect legumes, i.e. clover, beans, peas, etc.)
2. The legume plants produce chemicals called flavonoids to signal the Rhizobium.
3. The Rhizobium responds by producing a lipid-carbohydrate molecule
4. The lipid carbohydrate molecule stimulates the cells of the roots.
5. The cells of the roots divide rapidly. The cells from this rapid division swell to form a nodule. (A nodule is a modified root with a region of dividing cells - pericycle).
6. The bacteria have to enter the nodule.
   1. As a second response to the lipid carbohydrate molecule, root hairs with bacteria attached to them curl up, thus enclosing the bacteria.
   2. Enzymes produced by the bacteria digest the cell walls of the root hair cells, and the bacteria enter the cytoplasm.
   3. The plant then creates a tube (infection thread) along which the bacteria can grow. The thread stretches from the root hair cells to the center of the nodule. This delivers the bacteria to the nodule.
   4. The bacteria then move away from the thread and become covered by a special membrane made by the plant.
   5. After a few divisions, the bacteria lose their ability to divide and begin producing a nitrogen-fixing enzyme (called nitrogenase). This enzyme converts atmospheric nitrogen into ammonia - a compound that the plant is able to use.
   6. The bacteria are now called bacterioids and lose the ability to survive independent of the plant.

All plants require nitrogen to grow. They need nitrogen to assemble amino acids - these are the building blocks of proteins. These proteins contribute to plant growth and may also act as enzymes. Thus the overall result of increased nitrogen levels in soil is greener, healthier, and larger plants. Plants are only able to accept certain types of nitrogen. Sometimes this nitrogen is already present in the soil - it is made available through the work of soil bacteria. These bacteria, called nitrifying bacteria, break down organic matter into nitrogen compounds called nitrate (NO3-). This is then converted to ammonia by the plant - ammonia is used to make amino acids. Large quantities of atmospheric nitrogen exist in the soil as well, but plants are unable to utilize nitrogen in this form. Bacteria of the Rhizobia classification carry out biological nitrogen fixation, rearranging the molecules of nitrogen gas to form ammonia. They do this by binding the nitrogen to other elements.

         The legume we used was the field pea. Field peas derive 80% of their nitrogen from nitrogen-fixing bacteria. When grown as crops, they fix 155-175 lbs. of nitrogen per acre per year. This made it an excellent choice for an experimental plant, because such a large percent of its nitrogen is derived from bacteria. 80% is comparable to 50% in dry beans, 70% in chickpeas, and 90% in fababeans.

         The potential for nitrogen fixation is directly related to Rhizobia survival, the extent of effective nodulation and plant growth factors. A microorganism's ability to fix nitrogen is strongly influenced by soil conditions. These conditions include moisture, temperature, oxygen supply, nitrogen content, diseases, and insects. Any adverse soil condition or environmental stress that affects plant growth will slow down the nitrogen fixation process. Nitrogen fixation is also affected by the amount of available nitrogen in the soil. Legumes prefer to use available soil nitrogen before they use atmospheric nitrogen fixed by Rhizobia. Hence, high soil nitrogen levels reduce nitrogen fixation.

         Nitrogen fixation is more efficient then fertilization for many reasons. In the nitrogen fixation process, the nitrogen is converted to ammonia inside the plant. As a result, the plant does not need to expend energy to actively transport alternative forms of nitrogen into its roots. Another reason nitrogen fixation is superior to fertilization is because the rate of fixation is dependent on the availability of carbohydrates from leaves. Therefore the rate of fixation is synchronized with the rate of plant growth, making the process more efficient. Compared to fixation, fertilization is a relatively inefficient process. The nitrate supplied by fertilization can be easily washed away during a rainstorm. To utilize nitrogen from fertilizer, a plant must expend considerable energy to actively transport the nitrogen into the root cells and to convert it to a form that can be metabolized.

  Problem  

         How will Rhizobium Leguminosarum in the soil of pea plants affect their growth and development?

  Hypothesis  

         If one set of pea seeds is inoculated with Rhizobium Leguminosarum and then sown, then the plants that grow from those seeds will grow taller, faster, and healthier than peas that have not been inoculated.

  Procedure  

     A. Inoculate pea seeds with Rhizobium Leguminosarum
          1. Purchase Rhizobium inoculum
          2. Soak 192 pea seeds in water for 5 minutes
          3. Place 96 seeds in a separate container
          4. Pour some Rhizobium inoculum onto the seeds
          5. Shake the container until all the seeds are coated
              with the inoculum
     B. Plant pea seeds
          1. Find two large containers
          2. In each large container, place 8 smaller containers
              with holes in the bottom
          3. Fill the smaller containers ¾ full with Frank's seed
              starting soil
          4. Make 12 holes in the soil of each small container
              (holes should be 1 inch deep)
          5. Label one large container "With Bacteria". Label the
              other "No Bacteria"
          6. Sow the inoculated seed in the soil of the small
              containers in the "With Bacteria" group (place the
              seeds in the holes)
          7. Sow the non-inoculated seed in the soil of the small
              containers in the "No Bacteria" group (place the
              seeds in the holes)
          8. Pour water into the large container
          9. After five minutes, empty the large container
          10. Place the containers under fluorescent lights (unlit)
     C. Plant growth and measurement
          1. Water plants as needed
               a. Every morning, check to see if the soil is moist
               b. If the soil is becoming dry, pour water into the
                   large container
               c. After waiting for five minutes, empty the large
                   containers
          2. On Mondays, Wednesdays, and Fridays measure
              the plants
               a. Measure in cm.
               b. Measure from base of stem to apex of highest
                   leaf (Make sure plant stem is taut)
               c. Record results in log book
          3. Take pictures every Monday
          4. Make observations in the following areas (Do not
              record):
               a. greenness of leaves
               b. width of leaves
               c. diameter of stem
               d. number of leaves
               e. nodulation of roots
               f. plant weight

  Control  

         Plants grown from the non-inoculated seeds

  Constants  

• Type of plant (field pea)
• Unit of measurement
• Method of measurement
• Type of soil
• Intensity/type of light
• Height of light over plants
• Same small containers
• Air temperature
• Humidity
• Water source

  Independent Variable  

         Presence or absence of Rhizobium Leguminosarum in the root system of the plant

  Dependent Variable  

         Plant growth (height of plants)

  Data - Click for the enlarged graph  

  Pictures - Click to Enlarge  

  Analysis/Discussion  

         The plants that were inoculated with Rhizobium Leguminosarum grew taller, healthier, and faster. At the end of the project, we found nodules on the roots of the inoculated plants, but discovered no nodules on the roots of the non-inoculated plants. By the end of our project, the inoculated plants reached an average height of 17.99 cm. The plants without bacteria reached an average height of 16.8 cm. The inoculated plants were about 7% higher than the non-inoculated plants. With one exception, the plants with bacteria were taller than the plants without bacteria throughout the entire project. The pictures of our plants clearly show that the ones with bacteria looked much healthier than the ones without bacteria.

  Conclusions  

         We observed nodules on the group of plants that had been inoculated. The presence of these nodules represents a successful inoculation. The non-inoculated group did not have nodules, indicating that no Rhizobium Leguminosarum bacteria were present. In our hypothesis, we predicted that the plants with bacteria would grow taller, faster, and healthier than those plants without bacteria. Our experimental data supports this hypothesis. With one exception, the inoculated plants were consistently taller than the non-inoculated plants throughout the entire project. The plants with bacteria grew faster/taller/healthier because they were receiving more nitrogen than the plants without bacteria. The Rhizobium Leguminosarum provided the plants with this abundant source of nitrogen through the process of biological nitrogen fixation. In this process, the Rhizobium converts the nitrogen gas (N2) that lies between the soil particles to ammonia (NH4+). The pea plants use the ammonia to construct amino acids, which are later used in protein synthesis. To grow, plants need to synthesize proteins. Therefore, the plants with bacteria would always have enough ammonia to create the proteins that they required for growth. The non-inoculated plants, however, would rapidly consume the nitrogen in the soil and would then be unable to synthesize amino acids. Since no bacteria were present and no fertilizer was added, once the plants used their nitrogen, there was little left. With no nitrogen available to synthesize amino acids, a slowing of protein synthesis resulted, and, as a result, growth slowed as well. This is the reason why the inoculated plants grew taller than the non-inoculated plants. Nitrogen also contributes to vibrant, green plants. If one looks at the pictures, on will see that the inoculated plants were much healthier than the non-inoculated plants.

         We had many difficulties with our project. Our first few attempts to plant seeds failed. We tried different soils, different sowing techniques, and watered different amounts, but the results was always the same: rotten seeds. We then changed the seed we were using from bean to pea. The peas were sown with the inoculum in Frank's seed starting mixture. This time we were very successful. We concluded that there must have been a problem with the bean seeds.

         We made one mistake that must be mentioned. The plants with bacteria were labeled "No Bacteria" and the plants without bacteria were labeled "W/ Bacteria". It was not until the end of the project that we realized this error. When we noticed that the roots of the "No Bacteria" were covered with nodules and the roots of the "W/ Bacteria" had no nodules, we realized what our mistake must have been. The data has been rectified and all interpretation has been done as if the mistake did not occur.

         There are many different experiments that could branch from this one. We could expand the experiment to include different legumes (i.e. fababean, clover, etc.). We could try several different methods of inoculation to find the most effective method. We could experiment with different types of nitrogen fixing bacteria to find out if the pea plants were only compatible with certain strains of the Rhizobia classification (or which legume was compatible with which bacteria). We could vary soil conditions to see if that affected successful inoculation and nodulation. We could inoculate plants that were diseased or had insects to see if that had any affect on inoculation and nodulation. We could design an experiment to test whether the soil's nitrogen content had any affect on successful inoculation and nodulation. We could fertilize one set of plants and inoculate another set of plants, and see which one grew better. Many different projects could be inspired by our project.

         We made many mistakes that would be avoided if we did this project a second time. First, the project took too long. We wasted much time on failed experimentation. For example, in our first attempt we grew our own bacteria and sterilized our own soil. Before planting, we soaked the seeds in a nutrient broth that had been inoculated with the bacteria. We over-watered and allowed the soil to soak in stagnant water. In addition we did not put in enough soil. The result was rotten seeds. This first attempt took about a month, since we had to order the bacteria, wait for it to arrive, grow our own culture, then inoculate and plant the seeds. For our next attempt we bought some Frank's potting soil to plant the seeds in. We planned to inoculate the seedlings once they had sprouted by pouring the nutrient broth with the bacteria over the soil. Again we over-watered and allowed the soil to soak in stagnant water. Again the seeds rotted. For the third attempt we reused the soil from attempt number two, but this time mixed it with peat. We inoculated the seeds before they were sown using an inoculum provided by Mr. Waltman. This time three seeds sprouted. The rest rotted. For the fourth and final attempt, we used Frank's seed starting soil, inoculated the seeds with the inoculum before sowing, and used pea seeds instead of the bean seeds we had used previously. This time we had success. Almost all of the seeds sprouted, and there were no rotten seeds. But we made mistakes this time too. We positioned the trays next to each other, so it was possible for the bacteria to move to the other tray. Two different people measured, and their different techniques yielded different results. At times watering was erratic, and by the end, most of the plants were beginning to die. If we did this project again, we would do the following things differently:

• Do not be lazy about caring for plants
• Purchase seed starting soil - it's sterile
• Fill containers with soil - the soil should be no more than ½ inch from the top edge of the container
• Use inoculum on the seeds before sowing (Do not start your own culture)
• Water if the soil is becoming dry, and not at any other time (Bottom watering is best, but one must empty the bottom pan after the plants have had a chance to absorb the water - usu. about 5 minutes)
• Use the same type of large container for both groups (If you do not, another variable is created)
• Keep a large distance between the plants from the two groups (So it is not possible for the bacteria to get into the non bacteria group)
• Don't waste time (set up the experiment and finish it)
• Before planting, test the seeds capacity to germinate by placing them on a wet paper towel
• Make sure the labels on the containers are correct

  10 Things That Were Learned  

1. Excessive watering is harmful to plants and will rot the root systems.
   
2. Bottom watering is best, but the bottom tray must be emptied after the plants have absorbed the water.
   
3. If soil is baked for long periods of time, it loses many nutrients and its consistency (Baked soils are poor for plants).
   
4. The best type of soil for starting seeds is seed starting soil.
   
5. The best way to inoculate plants with Rhizobium Leguminosarum is to purchase an inoculum and coat the seeds with it.
   
6. Rhizobium Leguminosarum is a nitrogen-fixing bacterium that converts atmospheric nitrogen to ammonia. The plant uses the ammonia to make amino acids. In return, the plant supplies the bacteria with carbohydrates (which the bacteria use for energy.)
   
7. The techniques for growing bacteria in a petri dish
   
8. If left in a petri dish for too long, bacteria will draw all food from the agar and die, leaving a thin disc that once was the nutrient-filled agar.
   
9. Peas are monocots. As they grow, they produce tendrils that hook on to support. Peas eventually produce flowers, which self fertilize and begin to form pea pods (peas are fruits).
   
10. If you fail, change something and try again. (You're bound to get it right sooner or later)

  References Cited  

• Dazzo, Frank B. MSU Microbiology Facility. October 28, 1995. Available http://www.msu.edu/user/wingerdb/webpage/cmb/dazzo.htm (Dec. 17, 1997)

• Killpack, Scott C. and Daryl Buckholz. "Nitrogen in the Environment: Nitrogen Fixation." Department of Agronomy, University of Missouri- Columbia: WQ0261. October 1, 1993. Available http://muextension.missouri.edu/xplor/waterq/wq0261.htm

• "Nitrogen Fixation. "Microsoft Encarta 97 Encyclopedia". 1997 ed.

• NRI. "Nitrogen fixation: Nitrogen fixing plants?" May 12, 1995. Available http://lupin.csv.warwick.ac.uk/gene/pages/nitfix2.html (December 15, 1997)

• NRI. "Nitrogen fixation: Why is nitrogen important?" May 12, 1995. Available http://lupin.csv.warwick.ac.uk/gene/pages/nitfix1.html (December 15, 1997)

• "Soil Improvement with Legumes." Available http://www.gov.sk.ca/agfood/farmfact/scs03-2.htm (Dec. 17, 1997)
  
  
  
  
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