Nitrogen is approximately 78% (volume) of dry air. It is present in the protoplasm of living matter and the compounds contained in Nitrogen (Nitric Acid, Explosives, Cyanides, Fertilisers and Protein) are necessary to the continuation of life. Nitrogen is an essential constituent of Amino Acids that form Protein, which builds protoplasm. Although Nitrogen is about 78% (volume) of dry air this gaseous Nitrogen can?t be used by animals or plants. The Nitrogen must be ?Fixed? and turned into compounds such as Ammonia or Nitrates, which can be used. This is where Nitrogen Fixation comes in.
Nitrogen Fixation is the term to describe the reduction of atmospheric Nitrogen to Ammonia. Nitrogen Fixing bacteria live in the roots of Leguminous plants like beans peas and clover. The bacteria enter the plants through it?s root hairs and cause cells (cortical) of the root to proliferate which causes swelling called a Root Nodule. Vascular strands connect the nodule with vascular tissues in the main root. Bacteria rapidly multiply in the cells, fixing atmospheric Nitrogen which is then built up into Amino Acids and Proteins. The Amino Acid is then taken in by the plant to form plant tissue.
There are two main types of Nitrogen fixing bacteria, those that live free in the soil and those that live enclosed in the root nodules of leguminous plants. The free-living bacteria are species of Clostridium and Azotobacter. The se species are generally present in agriculture soils and use energy from decaying matter in the soil to fuel cell processes. The bacteria that live in the root nodules of leguminous plants are of the genus Rhizobium. Rhizobia can be found free living in the soil but can?t fix Nitrogen in this state and in turn the legume root cannot fix Nitrogen without Rhizobia. This is not the only way Nitrogen can be fixed. A certain amount of Nitrogen can be fixed by some blue-green algae and lightening but the soil bacteria previously mentioned perform the bulk of Nitrogen fixation. With all this in mind I have come up with the hypothesis: Clover seeds innoculated with Nitrogen Fixing bacteria will grow better than uninnoculated clover seeds when both grown in Nitrogen deficient conditions.
Method of Experiment
Test Tubes containing Nitrogen Deficient Agar
Rolled Cotton plug
Boiled cooled distilled water
Bleach 2% Hyper Chloride
Roots of Clover plant containing pink nodules
The clover seeds were purchased from a nursery and placed into a beaker containing bleach (2% Hyper Chloride) for five minutes to steralise them. The nodules were then rinsed in boiled, cooled distilled water. This process was repeated three times. The nodules were then place into test tubes containing Nitrogen deficient Agar using a steralised scalpel to transfer them. Rolled cotton plugs were then placed into the test tube and placed on a cool, well lit window.
Pink nodules were picked from the root of a clover plant and placed into a beaker containing bleach (2% Hyper Chloride) for five minutes to steralise them. The nodules were then rinsed in boiled, cooled distilled water. This process was repeated three times. The nodules were then transferred to a clean glass slide using steralised scalpels. The nodules were then squashed, also using a steralised scalpel, in a large drop of water. Steralised clover seeds were then transferred using a steralised scalpel into the squashed nodule mix and placed into test tubes containing Nitrogen Deficient Agar. The test tubes were then labelled and placed on a cool well-lit window with rolled cotton plugs in them.
Control Group – Mean stem length of clover plant over number of weeks
1 2 3 4 5 6 7 8 9 10
.75 1.62 2.01 3.11 3.56 3.84 3.48 3.35 3.31 3.3
Experimental Group ? Mean stem length of clover plant over number of weeks
1 2 3 4 5 6 7 8 9 10
.55 1.2 1.75 2.4 3.48 3.92 4.2 4.78 5.36 6.25
From these results you can see that the hypothesis: Clover seeds innoculated with Nitrogen Fixing bacteria will grow better than uninnoculated seeds when both grown in Nitrogen deficient conditions, was proved. In the first five weeks the control group grew slightly better than the experimental, but the experimental group looked healthier. It is after the sixth week that we see a dramatic difference in the two groups. The control group starts to decrease in size then level out and reach its maximum length. The experimental group increases rapidly to almost double the height of the control group. From these results we cannot see the maximum height of the experimental group, although given more time we would be able to see this.
Although the Hypothesis was proved, how can we make this experiment better? I think that if the squashed nodules were added to the clover seeds at the fifth week the development of the plant would be even more significant than before. The squashed nodules should be added at the fifth week just before the plant reaches its? peak. That way it gives the added nodules a week to start working before hand. The clover plant may grow to be bigger and better than the experimental plant, which could be beneficial.
The association between Legume ? Rhizobium is very specific, as I mentioned before Rhizobium cannot fix Nitrogen without legumes and vice versa nor can it fix Nitrogen with soybeans or lupins because it can?t nodulate with other species. I believe that once this hurdle is over come scientists will be able to produce Nitrogen Fixation with useful crops so that our productivity is greater.
In the future we may find out why the association between Legume ? Rhizobium is so complex and why they only nodulate with each other but for now we will just have to keep on experimenting to get it right.
Columbia New Illustrated Encyclopedia.,Vol 16, 1979, Rockville House, New York
A.H. Gibson Biological Nitrogen Fixation