Nitrogen Fixation

BY- K. Sai Manogna (MSIWM014)

Any natural or industrial process that allows free nitrogen (N2), a relatively inert gas abundant in the air, chemically combines with other components to form more reactive nitrogen compounds, such as ammonia, nitrates, or nitrites. 

Nitrogen does not react with other elements under ordinary conditions. However, in all fertile soils, in all living organisms, in many foodstuffs, in coal, and such naturally occurring chemicals as sodium nitrate and ammonia, nitrogenous compounds are contained. As DNA, nitrogen is also present in each living cell’s nucleus. 

 Role of nitrogen in nature: 

The growth of all organisms is dependent on the availability of mineral nutrients, and none is more crucial than nitrogen, which, as an integral component of proteins, nucleic acids, and other cellular constituents, is required in large quantities. In the earth’s atmosphere, there is an ample supply of nitrogen – approximately 79 percent in the form of N2 gas. However, since there is a triple bond between the two nitrogen atoms, N2 is unavailable for most species, rendering the molecule virtually inert. It must be ‘fixed’ (combined) in the form of ammonium (NH4) or nitrate (NO3) ions for nitrogen to be used for growth. The weathering of rocks releases these ions so slowly that fixed nitrogen availability has a marginal effect. Therefore, nitrogen is always the limiting factor for the growth and development of biomass in all habitats where there are an adequate climate and water availability to sustain life. 

In nearly all aspects of the availability of nitrogen and thus for life support on earth, microorganisms play a central role: 

Some bacteria can turn N2 into ammonia; they are either free-living or in symbiotic relationships with plants or other species (e.g., termites, protozoa). Other bacteria cause ammonia transformations to nitrate, and many bacteria and fungi degrade organic matter from nitrate to N2 or other nitrogen gases, releasing fixed nitrogen for reuse. These processes also lead to the cycle of nitrogen. 

Examples of nitrogen fixing bacteria

Biological Nitrogen Fixation Process 

Nitrogen cycle: The cycle of nitrogen is a repeating cycle process in which nitrogen travels through the soil, atmosphere, water, plants, animals, and bacteria, both living and non-living things. Nitrogen must change types in order to pass through the various parts of the cycle. Nitrogen occurs as a gas (N2) in the atmosphere, but it exists as nitrogen oxide, NO and nitrogen dioxide, NO2, in the soils and can be present in other forms when used as a fertilizer, such as ammonia, NH3, which can be further converted into another fertilizer, ammonium nitrate or NH4NO3. 

nitrogen cycle

The nitrogen cycle occurs in five steps: 

  1. Nitrogen fixation, 
  2. Mineralization, 
  3. Nitrification, 
  4. Immobilization, and 
  5. Denitrification. 

Microbes in the soil convert nitrogen gas (N2) into volatile ammonia (NH3) in this picture, so the volatilization is called the fixation process. Leaching is when specific nitrogen sources (such as nitrate or NO3) are dissolved in water, escaping from the soil and potentially polluting waterways. 

A. NITROGEN FIXATION: 

In this process, nitrogen moves into the soil from the atmosphere. A massive reservoir of nitrogen gas is in the earth’s atmosphere (N2). However, this nitrogen is not ‘available to plants because without transforming, the gaseous form cannot be used directly by plants. N2 must be converted by a mechanism called nitrogen fixation in order to be used by plants. 

1. In the atmosphere, fixation transforms nitrogen into which plants can absorb through their root systems.

2. When lightning gives the energy required for N2 to react with oxygen, creating nitrogen oxide, NO and nitrogen dioxide, NO2, a small amount of nitrogen can be fixed via rain or snow; these sources of nitrogen then enter the soil. 

3. By the industrial process that produces fertilizer, nitrogen may also be fixed. 

4. This method of fixation takes place under high pressure and heat, during which nitrogen(atmospheric) and hydrogen, combined to form ammonia (NH3); which can then be further processed for the development of ammonium nitrate (NH4NO3), a form of nitrogen which can be applied to the soil and used by plants. 

5. The majority of nitrogen fixation occurs naturally by bacteria in the soil. 

6. Some bacteria bind to plant roots and have a relationship with the symbiotic plant.

7. Through photosynthesis, the bacteria get energy, and in exchange, they fix nitrogen in a form that requires the plant. The fixed nitrogen is then transferred to other parts of the plant and used to shape the plant’s tissues to expand. 

8. Other bacteria live freely in soil or water, without this symbiotic relationship, and can fix nitrogen. These bacteria can also produce sources of nitrogen that species can use. 

B. MINERALIZATION: 

This process occurs in the soil from organic sources, such as manure or plant materials, nitrogen transfers to an inorganic source of nitrogen used by plants. The plant’s nutrients are gradually used up, and the plant dies and decomposes. In this stage of the nitrogen cycle, this becomes true. 

1. Mineralization occurs when bacteria, such as animal manure or decomposing plant or animal waste, operate on organic material, and begin to transform it into a nitrogen source that plants can use. 

2. All plants under cultivation, except legumes, obtain the nitrogen they need from the soil. 

3. Legumes get nitrogen through fixation that happens in their root nodules. 

4. NH3 is ammonia, the first source of nitrogen formed by the mineralization process. The NH3 in the soil then reacts to form ammonium, NH4, with water. 

5. This ammonium is kept in the soils and is accessible via the symbiotic nitrogen-fixing relationship mentioned above for use by plants that do not get nitrogen. 

C. NITRIFYING 

1. The third step, nitrification, also takes place in the soil. The ammonia formed during mineralization in the soils is converted into nitrites, NO2- and NO3-nitrates during nitrification. 

2. The plants and animals consuming the plants will use nitrates. 

3. In the soil, some bacteria can convert ammonia into nitrites. 

4. While nitrite is not usable by plants and animals directly, other bacteria may convert nitrites into nitrates, a form that is usable by plants and animals. 

5. For the bacteria involved in this process, this reaction provides energy. 

Nitrosomonas and Nitrobacter are the bacteria that help in fixing nitrogen. Nitrobacter transforms nitrites into nitrates; Nitrosomonas converts nitrites to ammonia. Only in the presence of oxygen can both forms of bacteria function. For plants, the nitrification process is essential as it creates an additional stash of usable nitrogen that can be consumed by the plants via their root systems. 

D. IMMOBILIZATION: 

Immobilization, often defined as the reverse of mineralization, is the fourth stage of the nitrogen cycle. Together these two processes regulate the amount of nitrogen in the soil. Microorganisms living in the soil, just like plants, require nitrogen as an energy source. 

1. When the residues of decomposing plants do not contain enough nitrogen, these soil microorganisms pull nitrogen from the soil. 

2. These nitrogen sources are no longer available to plants when microorganisms take in ammonium (NH4+) and nitrate (NO3−) and can cause nitrogen deficiency or a lack of nitrogen. 

3. Therefore, immobilization binds up nitrogen in microorganisms. 

4. Immobilization, however, is essential because it helps to regulate and balance the amount of nitrogen in microorganisms in the soils by binding it up or immobilizing the nitrogen. 

E. DENITRIFICATION: 

1. Nitrogen returns to the air in the fifth stage of the nitrogen cycle when bacteria transform nitrates to atmospheric nitrogen (N2) via denitrification. 

2. As the gaseous form of nitrogen travels into the atmosphere, it results in an overall loss of nitrogen from soils.

Not enough nitrogen in the soils makes plants hungry, while too much of a good thing can be harmful: plants and even livestock can be contaminated by excess nitrogen! The contamination of our water supplies by excess nitrogen and other nutrients is a significant concern, as the decomposition of dead algae blooms is suffocating marine life. Farmers and communities need to increase crop absorption of added nutrients and adequately manage the excess of animal manure.

RECOMBINANT DNA (RDNA) TECHNOLOGY

                   BY- ABHISHEKA G.(MSIWM013)

INTRODUCTION:

1.Recombinant DNA or RDNA technology is defined as the procedure of joining DNA molecules of two different species together and inserted into the host organism to produce a variety of new genetic combinations. This is also known as Genetic engineering.

2. The DNA fragments are selected from two different species and combined. This technique was developed by two scientists namely Boyer and Cohen in 1973.

3. The DNA molecule which is inserted into another DNA molecule is called a VECTOR. The recombinant vector is then introduced into a host cell where it replicates itself, and the new gene is produced. This is the basic principle behind Recombinant DNA technology.

TOOLS OF THE RECOMBINANT DNA TECHNOLOGY:

  1. Restriction endonucleases: These are used to cut DNA molecules at specific sequences into many smaller DNA fragments.
  2. Plasmids: These are extrachromosomal circular DNA present in the bacteria, which can replicate independently. During cloning, these plasmids carry drug resistance genes that are used for selection. Foreign DNA can be placed into a plasmid and it is replicated further.
  3. DNA ligase: This enzyme is used to join the two pieces of DNA together.
  4. Foreign DNA: This is also known as passenger DNA, which contains desired gene sequences.
  5. Vector: It is a vehicle used to insert the desired DNA into the host cell. Some of the vectors used are Plasmid DNA, Bacteriophage DNA, Yeast DNA, Viral DNA, Bacterial DNA, etc.

GOALS OF RDNA TECHNOLOGY:

  1. To isolate and characterize a gene or DNA from an organism.
  2. To eliminate undesirable phenotypic characters.
  3. To combine the needy and beneficial traits of two or more organisms.
  4. To make desired alterations in one or more isolated genes or DNA
  5. Inserting the altered genes or DNA into the host cell of another organism.
  6. To synthesize new genes using artificial methods.
  7. To alter the genome of the organism
  8. Understanding the diseases which transmit due to heredity.
  9. Understanding the treatment for heredity related disorders.
  10. To create new gene combinations.

PROCEDURE TO PREPARE RDNA:

1 Isolation of DNA from the organism: The cells are lysed using detergent mixtures, which creates pores in the plasma membrane. Then the mixture of cell contents is treated with protease and RNAase enzymes. The enzyme protease destroys the proteins present in the mixture and the enzyme RNAase destroys the RNA molecules present in the mixture. Then the mixture is centrifuged and the supernatant containing the DNA is transferred into a clean test tube and the DNA precipitated with the addition of ethanol.

2. Insertion of foreign genes into vectors: By using plasmid as a vector, isolated from the bacterial cell and treated with restriction enzymes and target DNA is obtained and it is placed into a vector to produce recombinant DNA.

3. Insertion of Recombinant DNA into host cell: The plasmid containing the foreign DNA is placed into a bacterial or host cell for multiplication.

4.Transformation: The vector is used as a vehicle to transport the gene to host cell, bacterium or other living cells are used as vectors. The vector is multiplied in the host cell and produces many identical copies, which are similar to both DNA and gene present in the DNA.

5. Cloning: After the division of the host cell the rDNA copies produced are transmitted to the progeny and further vector replication takes place in the progeny cell, with the continuous division of cells, a clone of identical host cells is formed. Each clone contains one or more copies of the rDNA molecule. Later the identical host cells are lysis and rDNA molecules are separated from the host cells.

APPLICATIONS OF RDNA TECHNOLOGY:

  1. This technology helps to grow crops which are resistant diseases and pesticides, crops of our choice, fruits, and flowers of attractive colors.
  2. This technique is employed in the production of artificial insulin and to deliver the drugs to target sites.
  3. Used in Molecular diagnosis of diseases.
  4. Used in Gene therapy.
  5. Employed in DNA fingerprinting.
  6. Used in the production of vaccines and pharmaceutical products.
  7. In the production of monoclonal antibodies.