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The effects of nitrogen-fixing bacteria on clover plants.

Updated on April 5, 2014


Encyclopaedia Britannica refers to nitrogen-fixing bacteria as microorganisms capable of transforming atmospheric nitrogen into fixed nitrogen, inorganic compounds usable by plants. There are various nitrogen fixing bacteria that exist in the soil.However, only a specific type of bacteria will attach itself to a specific plant nodules. This depends on whether the plant is leguminous, cereal grasses or blue/green algae. During this experiment Clover plants will be utilised and because this plant is considered to be a leguminous plant; the bacteria known as Rhizobium will be used

The Nitrogen Cycle

Nitrogen Fixation

  • Nitrogen Fixation is an anaerobic (without oxygen) process in which atmospheric nitrogen (N2) is reduced to ammonia (NH3 ). The bacteria known as rhizobium are responsible for converting atmospheric nitrogen into ammonia,which will be used by the clover plant in this experiment. Thus, converting nitrogen gas into a form that will be more accessible by the clover plants.
  • This process requires a specific enzyme, inorder to speed up the reaction. This enzyme is known as dinitrogenase.
  • Rhizobium bacteria present in the soil of the clover plant will require energy; inorder to convert nitrogen gas into ammonia. The rhizobium bacteria will require 16 moles of adenosine tri-phosphate (ATP) to reduce each mole of nitrogen. Rhizobium will access ATP by oxidizing the organic molecules.(Hubbell and Kidder,2009)
  • This equation summarises how ATP is used during the conversion of nitrogen gas to ammonia:N2 + 8H+ + 8e- + 16 ATP = 2NH3 + H2 + 16ADP + 16 Pi
  • The ammonia transferred from rhizobium bacteria to the clover plants will help to promote plant growth by meeting the clover plant's nutritional nitrogen needs for the synthesis of proteins, enzymes, nucleic acids, and chlorophyll.


Nitrification refers to the biological oxidation of ammonia with oxygen into nitrite followed by the oxidation of these nitrites (NO2-) into nitrates (NO3-).

The following equations will summarize the nitrification process:

2 NH4+ + 3 O2 → 2 NO2- + 2 H2O + 4 H+ (Nitrosomonas)

2 NO2- + 1 O2 → 2 NO3- (Nitrobacter, Nitrospina)

NH3 + O2 → NO2 + 3H+ + 2e

NO2 + H2O → NO3 + 2H+ + 2e


Denitrification refers to the process in which nitrate is converted to gaseous compounds (nitric oxide, nitrous oxide and N2) by microorganisms such as the rhizobium bacteria.

The sequenceusually involves the production of nitrite (NO2-) as an intermediate step for normal aerobic respiration, they use nitrate.

Experimental Images

The root of a clover plant which consists of rood nodules,produced by rhizobium
The root of a clover plant which consists of rood nodules,produced by rhizobium
A picture of a Wire Loop
A picture of a Wire Loop | Source
This picture illustrate the plate streaking technique.
This picture illustrate the plate streaking technique. | Source

Experimental Method

Method 1 –Isolation of Rhizobium bacteria

a Collect a plate containing mannitol yeast extract agar medium (MYEA) and make record of the date on the container.

NB: manitol yeast extract is newly isolated yeast isolated from soy-sauce mash.

( Onishi, Hiroshi, and Toshiyuki Suzuki. "Abstract." National Center for Biotechnology Information. U.S. National Library of Medicine, 19 Apr. 0006. Web. 04 Apr. 2014)

b Transfer several drops of ethanol to a sterile Petri dish using a Pasteur (dropping) pipette.

c Collect a clover plant. Rinse the soil off the roots under running water. Ensure that the root nodules are visible and not covered by dirt.

d Choose a length of root that has pink nodules and cut off a 1 cm portion using a scalpel. Hold the root by forceps and rinse off any more adhering soil using tap water.

e Place the clover plalnt’s root in a Petri dish of ethanol for 1-2 minutes to sterilise it.

f Use aseptic/ sterile technique from here onwards. Transfer 25 ml of sterile water to cover the base of a sterile Petri dish using a sterile Pasteur pipette. Keep the pipette sterile for later use by resting it under the lid of another sterile Petri dish.

g Sterilise forceps by dipping it into ethanol.Use the forceps to transfer the root to the sterile water to rinse off the ethanol .

h Repeat this twice more with fresh sterile water.

i Transfer a few drops of sterile water to a sterile Petri dish and add the portion of root using sterile forceps. Macerate (mash) the nodules with the forceps or a sterile glass rod to produce a milky fluid.

j The next step is to use a technique known as streak plate technique.

k Sterilise a wire loop by flaming, take a loopful of macerated nodules and make a streak on the medium. Flame the loop again.

l Secure the lids on the plates with adhesive tape.

m Incubate the plate at 20 – 25 °C for 3-4 days.

n After the time period has elapsed,study the colonies on the plate inorder to identify the rhizobium bacteria. Do not open the plate.

Nitrogeb fixation

What is nitrogen fixation?

See results

Method 2 - To discover the effects of rhizobium on clover plants

a Label each container with a different label

  • no additions (sand and plants)
  • inoculated ( rhizobium present) - no fertilizer (sand, bacteria and plants)
  • not inoculated (no rhizobium present) -fertilized (sand, fertilizer and plants)
  • inoculated (rhizobium present) - fertilized (sand, fertilizer, bacteria and plants)

b Put half of the sand (1 qt ) into a mixing bowl or container and add 1 teaspoon of fertilizer to the sand and mix thoroughly.

c Place 1 pint of the fertilized soil in each of pots labelled R& S.

Place 1 pint of the unfertilized soil in each of pots T & U.

Plant 5 clover seeds in each of the pots.

d Add the Rhizobium inoculum that was isolated; to the surface layer of the sand in pots U and S.

e Add moisture to the soils of each pot until water begins to run through the holes in the bottom of the container. Use distilled water.

f Place each of the pots in separate shallow dishes or trays.Place all containers in a warm area where;where sunlight can easily access the each container.Add water occasionally, in order to maintain moisture.

g Ensure that there are equal number of clover plant in each container.If any container contains less than 5 clover plant.Then remove the smallest plant from each container.

h Make and record measurements/observations at least two times per week.Repeat this procedure for nine to ten weeks.

i Measure and record the height of the aerial parts of the plants in each pot (cm). Determine the mean height (cm) for the plants in each pot. Record in the data table.Observe the appearance of the plants, including color, leaf size, leaf spacing, and any other observations and record them.

j Harvest the clover plants after nine to ten weeks. Wash the clover plant using distill water and avoid causing damage to each plan.Ensure that you remember the name(R,S,T,U) given to each plant.

k Describe the differences between root growth and root nodule formation of each clover plant.Place each clover plant into the sun and ensure that they are completely dry. Record the dry weight of the entire group of plants for each separate pot.

l Interpret the measured / observed results and reconcile them with your predictions as a written conclusion.

"National Teachers Enhancement Network: Lesson Plan." National Teachers Enhancement Network: Lesson Plan. N.p., n.d. Web. 04 Apr. 2014.

Summary of the Nitrogen cycle



  • Rhizobium are bacteria that helps to convert nitrogen gas into ammonium.
  • Rhizobium and clover plants share a mutual relationship
  • A deficiency in nitrogen compounds will cause the clover plants to grow less and the leaves will become yellow.
  • Ethanol helps to sterilize equipments used during the experiment
  • Nitrogen helps to develop important cell organelles.


When method 1 was completed, there were more than one bacteria found in the agar plate.However, the rhizobium bacteria were dominant. During, the experiment, ethanol played a significant role by sterilising the equipments. In method 2 a mutual relationship occurred between the rhizobium bacteria and clover plants.

The term mutualism refers to the close living relationships between two organisms, each organism gains an advantage from the presence of the other. After ten weeks had elapsed, the clover plants found in Pot S grew at a steady rate and the leaves of the plant were dark green. Pot S was introduced to an inoculated environment. Additionally, the wet mass of the clover plants in Pot S was considerably greater than the wet mass obtained from the clover plants in Pot U. The clover plants in pot U grew in an inoculated environment. This meant that Pot U did not consist of any fertilizer like Pot S.Pot U flourished at a moderate rate compared to Pot S. The leaves on the clover plants in Pot U were green. Though there was no fertilizer in Pot U, the clover plant still flourished because the rhizobium played a significant role, by converting nitrogen gas into ammonium.Thus,making nitrogen more accessible to the plants.Nitrogen will aid in the development in vital plant cell organelles such as chlorophyll.

The clover plants in Pot R were introduced to a non inoculated environment. This meant that there were no rhizobium bacteria introduced to the soil. Few of the clover plants were green.However,there were less green leaves present in Pot R compared to Pot S and U.In fact,quite a few of these leaves were yellow. This was a result of the absence of bacteria.Thus, the plants experience a deficiency in nitrogen compounds. The absence of rhizobium meant that the plant grew less. Pot T consisted of no fertilizer or rhizobium bacteria. It only consisted of clover plants and sand. The clover plant grew,but did not consist of much leaves when compared with the other pots.Most of its leaves were yellow compared to pot R and the wet mass was significantly less.

Expected Results

Average height of Pot T
Average height of Pot S
Average Height of Pot R
Average height of Pot U
January 28,2014
1 cm
January 29,2014
0.5 cm
February 8,2014
0.7 cm
1.8 cm
2.3 cm
February 11,2014
1.1 cm


In conclusion, rhizobium plays an important role in the development of leguminous plants such as clover plants. Rhizobium makes nitrogen easily available to plants.Nitrogen compounds obtained by the plants are then used to dictate the colour of each plants leaves as well as the development of important plant organelles.

Questions pertaining to experiment

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