Nitrogen Cycle Steps : Proteolysis, Ammonification, Nitrification Denitrification and Nitrogen Fixation and diagram

  Cycling of nitrogenous materials makes life on the Earth possible . The sequence of changes from free atmospheric nitrogen to fixed inorganic nitrogen, to simple organic nitrogen to complex nitrogeneous compounds in tissue of plants , animals and microorganisms and the eventual release of the nitrogen back to atmosphere is called Nitrogen cycle .

  Each process involves reduction or oxidation of nitrogen and each step is mediated by specific organisms having specific enzymes.

  The nitrogen cycle is due to the activities of decomposers and nitrogen bacteria. The nitrogen bacteria are grouped into three categories based on their roles as :
  • Nitrogen - fixing bacteria  
  • Nitrifying bacteria
  • Denitrifying bacteria

I). Nitrogen fixation :

   Thousands of tonnes of nitrogen is present in atmosphere, but this free N2 cannot be directly used by plants or animals.

Nitrogen fixation is a process in which atmospheric nitrogen is converted into ammonia which is then used in the biosynthesis of organic nitrogenous compounds, conversion of a limited amount of nitrogen to ammonia is done chemically during lighting strikes.

         N₂ + 3H₂➞ 2NH3

  The major amount of nitrogen fixation is biological and It is uniquely a procaryotic process.

  The nitrogen-fixing bacteria fix 70% of about 225 million metric tons of nitrogen annually. The cyanobacteria and bacteria that fix nitrogen are diverse and found in normal soils, deserts, hot-springs, marine as well as fresh water, Antartic regions, etc.

• Nitrogen fixation can be carried out under oxic and anoxic conditions. The nitrogen fixers can be grouped in two main groups:
(a) Non-symbiotic nitrogen fixers. (b) Symbiotic nitrogen fixers.

a). Non-symbiotic nitrogen fixing bacteria live freely and independently in the soil. The aerobic bacteria belonging to this group Include organisms like Azotobacter, Beijerinkia and cyanobacteria, and Trichodesmium.

  Cyanobacteria are procaryotic, free living algae that can fix nitrogen by obtaining hydrogen from hydrogen sulfide (H₂S) in sulfurous environments. Azotobacter are free-living soil bacterium and a heterotroph. The methylotrophic bacteria can also fix nitrogen when grown on methane, methanol or hydrogen containing substrates.

  Some genera of facultatively anaerobic bacteria that can carry out nitrogen fixation include Bacillus, Citrobacter, Enterobacter, Klebsiella, etc. Klebsiella pneumoniae can also fix nitrogen in the intestines of humans and other animals. The obligate anaerobes that carry out nitrogen fixation include the phototrophic bacteria belonging to the genera Rhodospirillum and chromatium, and other bacteria namely Clostridium, Desulfotomaculum, Desulfovibrio, etc.

(b) Symbiotic Nitrogen Fixers :
  Symbiotic nitrogen fixation is a process in which atmospheric nitrogen is fixed into ammonia by a mutualistic association between plants and bacteria, neither of which can fix nitrogen independently.

  The best studied nitrogen-fixing bacteria is Rhizobium the symbiont of roots of leguminuous plants. They can fix 150-200 kg of nitrogen per hectare of land annually.

  It is an endosymbiotic relationship between roots of leguminuous plants like clover, lupins, peas, beans, alfa-alfa and a nitrogen fixing bacterium Rhizobium. These bacteria establish themselves in the cells of root tissues of the host plant.

After developing infection thread on root hair, the infected plant grows abnormally with increased rate of growth forming nodule on the root system. In the nodule the rhizobla dwell and fix atmospheric nitrogen into NH4+ which is made available to plants.

These other symbiotic nitrogen fixing systems include actinomycetes and frankia-which can fix nitrogen symbiotically with many types of woody shrubs.

The genera Azospirillum fixes nitrogen association with grass/cereals. The cyanobacteria- Trichodesmium and Anabaena sp. fixes nitrogen in association with water fern Azolla.

  They are found in the pores of the leaves of Azolla and are used as biofertilizers in rice fields in Asia.

  The symbiotic nitrogen fixers has great nitrogen amount of nitrogen fixing ability and responsibe for large amount of nitrogen fixed globelly.

  The amount of nitrogen fixed by free living bacteria contributes to a small fraction of total nitrogen

Enzymes Involved in nitrogen fixation :
• The genetic information for nitrogen fixation is found in the 'nif' genes of nitrogen fxing microbes.
• The 'nif' genes encodes for the formation of an enzyme complex called nitrogenase system composed of nitrogenase and nitrogenase reductase.

The N, fixation reaction occurs as under :
N₂ + 8e¯ + 8H+ +  16MgATP ➞ 2NH3 + H₂+  16MgADP + 16Pi.

The nitrogenase system has two co-proteins, a MoFe cofactor containing molybedenum as well as iron, and Fe¯ protein containing only Iron.

The active nitrogenase is associated.with Fe, Mo - cofactor. At this site reduction of N₂ to NH3 occurs.

The electrons are transferred through ferredoxin or flavodoxin to nitrogenase (Fe-protein) reductase and then to nitrogenase.

  N₂ fixation requires large amount of ATP and H₂ production also occurs during this process.

  Only some strains of Rhizobium and Bradyrhizoblum have dehydrogenase to utilize the hydrogen. Other N₂ fixing bacteria evolve hydrogen gas and are often colonized by hydrogen oxidizing Acinetobacter strains.

Nitrogenase is very sensitive to oxygen and is irreversibly Inactivated by even low concentration of O₂.

Nitrogen fixation Is therefore restricted to habitats in which nitrogenase is protected from exposure to molecular to molecular oxygen.

Both symblotic and  non symblotic N₂ fixers can be used as blofertillizer to increase soil fertility.

The product of N₂ fixation is ammonia, which is immediately incorporated into organic matter as an amine (glutamine). These amino N atoms are converted into amino acids and proteins, nucleic acid and other biomolecules in plants, animals and microorganisms.

The N₂ cycle continues with the degradation of these molecules into NH4+ within many microbes through many pathways including proteolysis.

II). Proteolysis :

Plants use ammonia produced by symbiotic nitrogen fixers, nonsymbiotic nitrogen fixers, and ammonia available through assimilatory reduction of nitrates to synthesize amino acids and eventually plant proteins.

  When plants are consumed by animals the plant protein are converted to animal proteins. This immobilized nitrogen in animal and plant proteins and other nitrogenous compounds can be released only when plants and animals die.

These proteins and other nitrogenous compounds are decomposed in the soil.

The process of enzymatic breakdown of proteins is called proteolysis.

Proteolysis is carried out by microbes which produce extracellular enzymes called Proteases.

The proteins are converted to smaller molecules called peptides, which are further decomposed to amino acid by enzyme peptidase.

Proteinases are produced by many fungi and bacteria including Clostridium, Bacillus, Pseudomonas, Proteus, Aspergillus, Mucor, spp. etc. Peptidases are produced by many bacterial genera. The process is also known as dissimilation of organic nitrogen.

III). Ammonification or Amino Acid Degradation :

The end product of proteolysis are amino acids which are further degraded by soil microorganisms for use as nutrients.

• Ammonification is the process in which release of ammonia takes place from complex organic nitrogenous compounds. It usually occurs under aerobic condition. The microorganisms responsible are Peus, Bacillus, Microccus, etc.

• The ammonia produced has different fates :
(1) As it is volatile, it may leave the soil.
(2) It may be solubilized in water and ammonium lons are produced.
(3) Ammonium lons can be utilized both by plants and microorganisms.
(4) It may be oxidized to nitrates by a process called nitrification.

Putrefication is the anaerobic degradation of amino acid resulting into formation of amines. This is mainly carried out by anaerobic bacterlum belonging to genus Clostridium.

IV). Nitrification :

This is a two step process carried out by chemolithotrophs.

The first step is sometimes described as Nitrosofication where NH4+ is first oxidized to nitrite by bacterial genera Nitrosomonas, Nitrosobacter, Nitrosolobus, Nitrosococcus, etc.

The reaction occurs as follows:
NH4+ + O ➞ NO₂¯ + H₂O + 2H+ + energy
* NH4+ ammonium lons
* NO₂¯ Nitrite

These bacteria are chemolithotrophic, gram negative, aerobic, motile, rod-shaped bacteria, sensitive to acidity.

The second step described as Nitrification is carried out by nitrite oxidizing bacteria as under :
NO₂¯ + O ➞ NO3¯(nitrate) + energy

The nitrite produced in the first step is toxic to plante the nitrification of nitrite to nitrate is very useful for agriculture.

Microorganisms involved in Nitrification:
Bacterial genera Nitrobacter, Nitrospira and Nitrococcus. They are chemolithotrophic gram negative, nonmotile bacteria, rods, sensitive alkaline conditions.
  In addition Nitrosomonas eutropha has been found to oxidize ammonium ion anaerobically to nitrite in a denitrification related reaction.

V). Assimilatory nitrate reduction :

The production of nitrate by nitrification is important because it can be reduced and incorporated into organic nitrogen by plants.

The use of nitrate as a source to synthesize organic nitrogen is called assimilatory nitrate reduction.

Many heterotrophic bacteria can also reduce nitrate to ammonia to organic nitrogen by assimilatory reduction.
NO3¯ + 8e¯ + 9H+ ➞ NH3 + 3H₂0

The ammonia formed is used for the synthesis of amino acids ➞ proteins ➞ other nitrogenous compounds. This process occurs under aerobic condition, in water-logged soil. The oxygen of nitrates serves as an acceptor o electrons and hydrogen.

VI). Denitrification :

In this process nitrates are reduced to nitrous oxide (N₂O) or molecular nitrogen by certain soil bacteria. The process occurs under condition of oxygen limitation.  

The sequence of reactions are as follows:
2NO3¯➞ 2NO₂¯➞  2NO ➞ N₂O  ➞ N₂↑
Nitrate ➞ Nitrite ➞ Nitric Oxide ➞ Nitrous Oxide ➞ free Nitrogen

This process results in a net loss of N₂ from soil into the atmosphere.

The microorganisms responsible for Denitrification:
Achromobacter, Agrobacterium, Alcaligenes, Bacillus, Micrococcus, Pseudomonas, Thiobacillus, Vibrio etc.

Several anaerobic bacteria in soil carry out dissimilative nitrate reduction in which nitrate is reduced to ammonia as follows:
  NO3¯+ H₂ ➞ NH3 ➞ N₂O

The process is enhanced by :
(1) Presence of organic matter
(2) Temperature between  
      25-60°C.
(3) Neutral to alkaline pH.
(4) Anoxic condition.

The major products of denitrification include N₂ gas and nitrous oxide, although nitrite also can accumulate. Nitrite is of environmental concern because it can contribute to the formation of carcinogenic nitrosamines.

Finally nitrate can be transformed to ammonia in dissimilatory reduction by a variety of bacteria including Geobacter metallireducers, Desulfovibrio spp., Clostridium spp., etc.

A recently Identified form of nitrogen conversion is called the anammox reaction (anoxic ammonium oxidation process). In this process anaerobic, chemolithotrophs use ammonium ion as electron donor and nitrite as the terminal electron acceptor.

The marine planctomycete bacteria oxidize large amounts of NH4+ ion to N₂ through anammox reaction.

Denitrification removes nitrogen from soil, thereby reducing fertility of soil.