FREE ESSAY ON NITROGEN FIXING

NITROGEN FIXING

Clover, growth rate, inoculation with Nitrogen fixing bacteria in Nitrogen deficient
conditions
Nitrogen Fixation was proved to increase the growth of clover plants over a ten-week
experiment in Nitrogen deficient conditions. The Hypothesis was proved correct with no
difficulties encountered.
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
Materials
Clover Seeds 
Test Tubes containing Nitrogen Deficient Agar
Rolled Cotton plug
Beakers
Boiled cooled distilled water
Bleach 2% Hyper Chloride
Scalpels
Microscopic Slides
Roots of Clover plant containing pink nodules
Bunsen Burner
Control Group
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.
Experimental Group
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. 
Results
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
After Five weeks the Experimental Group looked a lot healthier. It's leaves were larger,
more numerous and rich in colour. The Control Group's leaves were much paler and less
numerous.
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. 
We know that Nitrogen fixation improves legume plant quality but can it improve the
quality of more useful plants such as crops? Alan H. Gibson from the CSRIO Division of
Plant Industry believes not.
" my colleagues and I believe this to be most unlikely due to the stringency of the
conditions necessary for these genes to be expressed."
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. 
Bibliography
Roberts, M, Reiss,M and G, Monger. 1993, Biology Principles and Processes, Thomas Nelson
and Sons, Edinborough
Columbia New Illustrated Encyclopedia.,Vol 16, 1979, Rockville House, New York
A.H. Gibson Biological Nitrogen Fixation