Species, Nitrogen Fixation, Biofertilizer and Culture


Rhizobium are a group of Gram-negative soil bacteria that are well known for their symbiotic relationship with various leguminous (soybeans, alfalfa etc).

There are different types of Rhizobium that are categorized on the basis of the rate of growth and the type of plant they are associated with.

Some species of Rhizobium include:


  • R. leguminosarum
  • R. alamii
  • R. lentis
  • R. japonicum
  • R. metallidurans
  • R. smilacinae
  • R. phaseoli
  • R. trifolii


Along with Bradyrhizobium, Sinorhizobium, Mesorhizobium, Azorhizobium, and Allorhizobium, Rhizobium is a soil Rhizobia, which means that it consists of bacteria with the ability to fix nitrogen. As such, it presents a significant advantage to the plants it infects by contributing to their growth and development.

It's classified as follows:


  • Kingdom: Bacteria - Like other bacteria, Rhizobium cells lack membrane-bound organelles. They also have ribosome, cytoplasm and flagella. 
  • Phylum: Proteobacteria - Phylum proteobacteria includes Gram-negative bacteria including those responsible for fixing nitrogen.
  • Class: Alphaproteobacteria - Class Alphaproteobacteria  is composed of a wide range of Gram-negative with varying life characteristics.
  • Order: Rhizobiales - This is an order of Gram-negative Alphaproteobacteria that form a symbiotic relationship with plant roots (where they fix nitrogen).
  • Family: Rhizobiaceae - Along with such genera as Agrobacterium, genus Rhizobium makes up the family Rhizobiaceae. While some in this family have been shown to negatively affect plant development, Rhizobium  (a member of Rhizobia) contribute to plant nutrition through nitrogen-fixation.
  • Species: Includes such species as Rhizobium leguminosarum and Rhizobium lentil.


In addition to this classification, Rhizobium bacteria are also categorized based on the species of legume that they nodulate. This type of grouping is known as cross-inoculation.

Based on studies on a wide variety of legumes, it became evident that not all Rhizobia are capable of nodulating all types of legumes. This resulted in a need to group Rhizobium into appropriate groups (cross-inoculation groups) which are simply groups of legumes that given species of Rhizobium nodulate.

The cross-inoculation groups include:


  • Clover groups - R. trifolii infects and nodulates plants of genus Trifolium (clovers/trefoil)
  • Alfalfa groups - R. meliloti infects and nodulates the roots of medicago, melilotus and medicago
  • Bean group - R. phaseoli infects and nodulates plants of genus Phaseolus (e.g. beans)
  • Lupine group - R. lupine nodulates lupines and serradella (Ornithopus)
  • Pea group - R. leguminosarum infects and nodulates pea, sweet pea, lentil, and vetch
  • Soybean group - R. japonicum nodulates Glycine such as soybean
  • Cowpea group - Rhizobium sp. nodulates cowpea, pegionpea, lespedza, groundnut and kudz among a few others. 


* The word Rhizobium comes from the Greek words: "rhiza" which refers to root, and "bios" which refers to life.


Characteristics (Rhizobium Leguminosarum)

Discovered and described in 1889, R. leguminosarum is the type species of Rhizobium in the same way Rhizobium has been the type genus of family Rhizobiaceae. As such, it can be viewed as a representative of the genus (Rhizobium).

Some of the characteristics of the bacteria include:


  • They appear as elongated rods when viewed under the microscope 
  • Like a number of other bacteria, Rhizobium leguminosarum do not form spores in their life cycle
  • They posses several flagella on their polar end. This allows them to move from one location to another
  • They are aerobic. As such, they need oxygen for respiratory purposes
  • There are various strains of the bacteria some of which have granules 
  • They are Gram-negative bacteria 
  • Although they can tolerate higher temperatures of about 38 degrees Celsius, Rhizobium leguminosarum ideally grow in temperatures of between 20 and 28 degrees Celsius
  • Apart from various types of carbohydrates, the bacteria also uses nitrates and nitrite, ammonium salts and various amino acids among others for development


There are different strains which include:


  • Rhizobium leguminosarum biovar viciae 3841
  • Rhizobium leguminosarum USDA2370T
  • Rhizobium pisi DSM30132T
  • Rhizobium fabae CCBAU33202T

Infection (Penetration)

Rhizobium species like R. leguminosarum can be found in soil. However, the root of leguminous plants (lentil, sweetpea etc) is their primary habitat. In the soil, various leguminous plants release various exudates (dicarboxylic acids etc) that attract Rhizobium species.

Flavanoids have also been shown to play an important role attracting the bacteria given that they are easily absorbed through the membrane of the organisms (passively). Once the bacteria detect these chemicals, they actively swim towards and attach to the legume root. 

In addition to attracting the bacteria, these chemicals (flavanoids in particular) also play an important role of activating genes involved in producing Nod factors. Here, then, attraction to the legume roots is followed by transcription of Nod genes in preparation of the symbiotic relationship.

For the plant, Nod factors stimulate the branching of root hair, hydrolysis of the cell wall as well as deformation of the cell wall. Having attracted the bacteria through the exudates, the changes in the plant roots make it easy for the organism to enter the cells of the root hair for symbiosis. 

* When the bacteria comes into contact with the root hair, they cause the plasma membrane of the cells to invaginate. As the bacteria penetrates the cell, the plant produces new cell wall material at the site to not only cover the bacteria, but also allowing them to enter deeper into the root hairs. 


Once the bacteria infects the cells of the root hair, the symbiotic process may produce the following types of nodules:


  • Determinate nodules - Determinate are found in plants like soybeans, are spherical in shape with large lenticels. Compared to indeterminate nodules, determinate nodules are formed once the tip of the root hairs start growing. However, they lack the persistent meristem found in indeterminate nodules.
  • Indeterminate nodules - Compared to determinate nodules, indeterminate nodules are cylindrical in shape and frequently branched. They are often found in plants like peas and alfalfa and start developing even before the growth of the root tip. 

* Whereas indeterminate nodules are found in temperate regions, determinate nodules are commonly formed in tropical and sub-tropical areas. 

* Determinate nodules produce ureide products while indeterminate nodules amide products. 

Nitrogen Fixation

Nitrogen fixation in the nodules begins when the nodules fully mature. Here, it is worth noting that nitrogen fixation involves the conversion of atmospheric nitrogen into organic compounds (particularly ammonia) that can be used for plant development.

This process requires two important genes (nif and fix). These genes play an important role of producing several crucial enzymes that are involved in the nitrogen fixation. 

The process requires the following:


  • Enzymes (dinitrogenase and dinitrogenase reductase)
  • ATP energy 
  • Atmospheric Nitrogen (Nitrogen molecules)



During nitrogen fixation, the enzyme nitrogenase is involved in the breaking of the bonds that hold Nitrogen atoms together (covalent bonds). In their atmospheric state, nitrogen molecules are non-reactive given that they are bound by covalent bonds. By breaking this bond, the Nitrogen atoms are free to form bonds with other atoms.

This process (breaking down the covalent bonds) requires a lot of energy. Given that Rhizobium bacteria are not capable of making their own food for energy, they rely on the plant (in the rhizospere) to provide sources of energy.

By using energy sources from the plant, the bacteria gains sufficient energy that makes it possible for the enzymes to break down Nitrogen molecules into Nitrogen atoms. 


* Nitrogenase enzyme is oxygen sensitive. However, high metabolic activity of the bacteria as well as a diffusion barrier developed at the nodule periphery help protect the enzyme from the high level of oxygen.


Nitrogen fixation in the nodules takes place while the Nitrogen molecules are attached to the enzyme. While the nitrogen is bound to the enzyme, electrons provided by ferredoxin make it possible for the Iron protein of the enzyme to be reduced. This protein then binds to the ATP, which in turn causes molybdenum-iron protein (also a  component of the enzyme) to be reduced.

This reduction provides electrons required to break down the Nitrogen molecules and produce a compound known as Diimide ((NH)2). The process is repeated two more times which further reduces the Diimide into two ammonia molecules. 



The process is represented as follows:


N2 + 8H + 8e- + 16Mg-ATP                         2NH3 + H2 + 16MgADP + 16Pi


* The relationship between leguminous plants and Rhizobium bacteria is referred to as a symbiotic relationship because the bacteria and the plant benefit each other. While the plant’s rhizosphere provides shelter and a source of energy for the bacteria, the bacteria converts atmospheric nitrogen to ammonia which is required for proper growth and development of the plant. 


Nitrogen is one of the most important elements in nature given that it is used to make various products that plants require for their development. For instance, using such products as ammonia and nitrates that are made from nitrogen, plants are able to form the protein they need for their development.

While various chemical fertilizers are successfully used to increase yields, they are also expensive and tend to pollute the environment. Given that Rhizobium bacteria have a good and beneficial relationship with various leguminous plants, there has been increased interest to use them as biofertilizers. 

In small scale scenarios, the inoculation of Rhizobium in various cereal grains has been shown to help increase yields. This was also shown to result in an increased uptake of nitrogen thus benefiting the plants.

On a large scale, production of the inoculants begins with identifying the most effective strain of bacteria through host-specificity. Here, research helps identify the most effective strain of Rhizobium that is then cultured for large scale production.

During mass culture production, the bacteria is cultured in large flasks composed of such carbohydrates as mannitol, arabinose and sucrose among others. This helps produce fertilizer products that have been shown to help enhance photosynthesis and thus yields such plants as banana and rice among others significantly. 

* Biofertilizer presents a significant advantage in that they not only increase yields for farmers, but are also safe and do not negatively affect the soil and environment they are used in. Studies in different parts of the world such as Sudan have found this method to be particularly beneficial for a variety of crops.


Sample Preparation 


  • Carefully uproot a leguminous plant (sweetpeas, beans etc)
  • Gently wash the roots using sterilized water to remove any soil, mud etc.
  • Using a clean knife/blade, carefully remove/detach the nodules (which are visible to the naked eye) from the roots and place them in a container with clear water
  • After washing the nodules, place them in a container with a solution of merculic chloride for a few minutes - this helps disinfect and remove any microorganism on the surface of the nodule
  • Wash the nodules using alcohol and then wash with clean water several times
  • Place the nodules in a test-tube and add a few drops of water (distilled water)
  • Using a glass rod, crush the nodules to release the bacteria into the water


Culture Procedure 


  • Gently pour mannitol-agar medium into 3 sterile test-tubes
  • Add a few drops of the sample (rhizobium water) into the test-tubes using sterilized droppers
  • Mix the contents thoroughly
  • Pour the contents into different Petri dishes and incubate at 45 degrees Celsius
  • Turn the Petri dish when the medium solidifies
  • Allow the culture to incubate for about 3 days


* Sample may also be collected from soil surrounding the roots of leguminous plant for comparison

Gram Staining

Using a sample from the culture, Gram-staining involves the following steps:


  • Fix the bacteria using methanol. Having placed the sample at the central part of a clean slide, add a few drops of methanol to fix. This helps preserve the morphology of the bacteria
  • Allow the slide to dry and pour the primary stain on the sample (crystal violet) - Allow to stand for a few minutes
  • Add a few drops of the mordant (Iodine solution) 
  • Decolorize the sample using ethanol/acetone gently 
  • Pour safranin on the sample (counter-stain) for a few minutes 
  • Wash gently with water to remove any excess counter-stain
  • Mount and view under the microscope 


The bacteria will appear as red/pink rods under the microscope with several flagella on one polar end.

When measured, they are about 0.5 to 0.9um in width and 1.2 to 3.0 um length.

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H. Panda. (2011). Manufacture of Biofertilizer and Organic Farming. 
Frédéric Zakhia and Philippe De Lajudie (2001). Taxonomy of rhizobia 

Niir Board (2004). The Complete Technology Book On Bio-Fertilizer And Organic Farming 

Shefali Poonia. (2011). RHIZOBIUM: A Natural Biofertilizer. International Journal of Engineering and Management Research Vol. 1, Issue-1, November-2011.  Links




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