Nitrogen Cycle

NITROGEN CYCLE-An Important Biogeochemical cycle in Biosphere

Nitrogen forms the main bulk of the atmosphere (78%) as well as the biological systems. Various nitrogenous compounds, e.g. proteins, enzymes, chlorophylls, nucleic acids, etc. play viral roles in the life processes of organisms. The atmospheric nitrogen is chemically inert and is not directly taken by most of the living organisms. The latter, therefore, depend on a source of combined nitrogen or organic nitrogen compounds for their growth. They obtain nitrogenous compounds from soil and organic nitrogen compounds for their growth. They obtain nitrogenous compounds from soil and convert them into essential bimolecular needed for their healthy development. In addition, a part of the great reservoir of the atmospheric nitrogen is converted into an organic form by certain free living microorganism and by plant microorganism association which make it available to the plants. Animals obtain it from plants. The percentage of nitrogen in the atmosphere remains constant by the operation of a nitrogen cycle in nature. Nitrogen is continually entering in the air by the action of denitrifying bacteria and continually returning to the cycle through the action of nitrogen fixing microorganism, lightening, and industrial production of artificial fertilizer. These sequences of changes from free atmospheric nitrogen to fixed inorganic nitrogen, to simple organic compounds, to complex organic compounds in the tissues of microorganisms, plants and animals, and the eventual release of this nitrogen back to atmospheric nitrogen is dealt under the ‘nitrogen cycle’

N in the atmosphere: 3800,000 x 10⁹ ton

N in the plant biomass: 12 x 10⁹ ton

N in the dead organic matter (on land): 300 x 10⁹ ton

N in the plant biomass (in oceans): 0.3 x 10⁹ ton

N in the organic matter (in oceans): 550 x 10⁹ ton

1. Dinitrogen Fixation:

Only prokaryotes are capable of dinitrogen fixation.

A. Free living N₂ fixing bacteria:

Aerobic: Azatobacter, Methane oxidizing methylotrophs, Cyanobacteria

Microaerophillic: Azospirillum, Rhizobium

Facultative anaerobes: Enterobacter, Klebseilla

Anaerobic: Clostridium, Phototrophs, Desulfovibrio

B. N₂ fixing symbiotic association:

Symbisis: Rhizobium, Bradyrhizobium, Frankia

Rhizosphere: Azospirillum, Azatobacter paspali, Klebseilla

Figure 1: Schematic diagram of Nitrogen cycle

N₂ fixing symbiotic association between root nodules and rhizobia:

It is a very important symbiotic association between leguminous plants with Rhizobium. Two more genera are involved in N₂ fixation are Azorhizobium and Bradyrhizobium. Rhizobium are fast growing organism found in root nodules of Alfalfa, Peas, Clover, other leguminous etc. While Bradyrhizobium are slow growing one found in association with soyabeans, lupine, cowpeas, tropical leguminous plants etc. The Azorhizobium grow with atmospheric nitrogen in its free living state.

Naturally, Rhizobia are free living organisms in soil. They are not directly involved in atmospheric nitrogen fixation. However they can invade root hairs, initiate the formation of a nodule on specific plant and develop nitrogen fixing activity under appropriate condition. The nodule formation is a complex sequence of interactions between rhizobia and plant roots. At the starting of nodule formation, plants secrete flavonoids and isoflavonoids which are attracts particular group of rhizobia by chemotaxis. These chemicals induce the expression of a number of nodulation (nod) genes in rhizobia. The gene codes for the production of enzymes which involved in the biosynthesis of species-specific substituted lipopolysaccharides called nod factors. The factor signals roots and elicit the curling of plant root hairs and division of meristematic cells leading to the formation of nodules.

At the beginning, both Rhizobium and Bradyrhizobium species are attracted by amino acids and dicarboxylic acids presents in the root exudates to their specific legume plants. During nodulation process, tryptophan secreted plant root is metabolized to indole acetic acid (IAA) by the rhizobia. The IAA initiates the curling of root hairs which grow around the rhizobia. Then the bacteria penetrate the soft tissue of plant root developing an infection tube (thread) that is surrounded by cell membrane and cellulosic wall. The infection thread extends to the root cortex region. Within the infection tissue, rhizobia multiply forming unusually shaped and sometimes grossly enlarged cells called bacteroids. The bacteroids produce and contain active nitrogenase enzyme which actually takes part in nitrogen fixation.

Figure 2: Schematic diagram of Nitrogen fixation in Bacteroids

The nitrogenase enzyme system catalyzes the reduction of molecular nitrogen to ammonia. The enzyme system is a complex of dinitrogenase reductase (Fe protein) and dinitrogenase (MoFe protein). Electrons are initially transferred to the dinitrogenase reductase (Fe₄S₄ center), They are then transferred to the P clusters (Fe₄S₄) of the dinitrogenase protein. The P clusters pass the electrons to the iron-molybdenum cofactros (FeMoco; Fe₇S₉Mo-homocitrate) of the dinitrogenase and then on to N₂-H₂ is also evolved in the reaction. Nitrogen fixation brought about by nitrogenase producing bacteria converts atmospheric nitrogen to fixed forms of nitrogen (NH₃, which at physiological pH occurs as NH₄⁺) that can be used by other microorganism, plants and animals.

2. Ammonification:

Ammonia released during aerobic and anaerobic decomposition of organic matter is known as ammonification. However the ammonia released by the process is rapidly recycled by plants and microorganisms. Losses of NH3 by vaporization amount to 15% of the total nitrogen loss, 85% being loss from denitrification. Under anaerobic conditions, NH4+ is a stable compound. Two different steps are involved in ammonification: (a) Proteolysis and (b) Amino acid degradation (Deamination).

(a) Proteolysis: Breakdown of protein into its simpler forms is called proteolysis. A number of bacterial species e.g. Clostridium spp. Pseudomonas, Proteins, Bacillus, and soil actinomycetes, and many fungi are extremely proteolytic. They secrete extracellular enzyme, namely, ‘Proteases’ that convert the protein to smaller unites (peptides) which are then attacked by other proteolytic enzyme, namely ‘peptidases’ resulting ultimately in the release of amino acids. The overall process can be summarized in the form of reactions as follows.

Proteins=/proteses/=Peptides=/peptidase/=Amino acids

(b) Amino acid degradation (Deamination): The release of NH₃ from amino acids occurs by different types of deamination.

1. Oxidative deamination:

Glutamate ® 2-oxoglutamate

Glutamate dehydrogenase

This process is most important in amino-acid metabolism.

2. Desaturative deamination:

Aspartic acid ® fumarate + NH₃

aspartase

3. Hydrolytic deamination:

Urea ® NH₃ + CO₂

Urease

Urease is repressed by NH₄⁺, but also exists as constitutive enzyme, e.g. Proteus spp. and in Sporosarcina ureae.

At neutral pH, little free NH₃ is present and the ionized from NH₄⁺ prevails. Microoganisms dispose of several reactions for primary NH₄⁺ assimilation.

1. NH₄⁺ + 2-oxoglutarate ® glutamate

Glutamate dehydrogenase

2. NH₄⁺ + Pyruvate ® alanine

Alanine dehydrogenase

3. NH₄⁺ + glutamate ® glutamine

Glutamate synthetase

Glutamine synthetase has a high affinity to NH₄⁺ and operates at extremely low NH₄⁺concentration (less than 1mM).

3. Nitrification:

Nitrification is the aerobic process in which ammonia is oxidized to nitrite and nitrite to nitrate by the chemolithotrophic nitrifying bacteria. The nitrification process occurs in two stages.

A. Ammonia is first oxidized to nitrite via hydroxylamine. The reaction is catalyzed by oxygenase and hydroxylamine reductase.

NH₃ + O₂ + [O] ® NH₄OH

Ammonia oxygenase hydroxylamine

NH₄OH + O₂® NO₂^- + ATPs

Hydroxylamine Hydroxylamine reductase nitrite

Overall reaction: NH₄⁺ + 3/2O₂ ® NO₂^- + 2H⁺ +H₂O +276KJ

This first step occurs best at high pH values as enzymes involved prefer non-ionized NH₃ form. The bacteria involve in this step are; Nitrosomonas, Nitrosospira, Nitrosolobus, Nitrosovibrio and Nitrosococcus.

B. In the second step, nitrite is oxidized to nitrate, a stable nitrogen oxide form.

NO₂^-+ 1/2O₂ ® NO₃^- + 73KJ

The bacteria involve in this step are; Nitrobacter, Nitrospina, and Nitrococcus. In addition certain fungi e.g., Cephalosporium, Aspergillus and Penicillium have been reported able to carry out nitrification were discovered to be a biological process by Schloesing and Muntz (1877); Winogradsky isolated the bacteria responsible for biological nitrification in 1890.

The whole process is strict aerobic process and doesn't occur at redox values lower than +200mV (E_o). The optimum pH for nitrification is at neutral pH or slightly alkaline pH condition (pH 7-8). The nitrate is highly soluble and leached out easily. Because of this property, it is a disadvantage for agriculture.

4. Denitrification:

Denitrification refers to the conversion of nitrate (nitrite) into dinitrogen and gaseous oxides of nitrogen (NO, N₂O) by bacterial nitrate respiration. The process occurs under anoxic conditions and is performed by essentially aerobic bacteria. Nitrate serves as an alternative electron acceptor in the absence of molecular oxygen. Denitrification has attracted much interest for several reasons. 1) Loss of fertilizer nitrogen means a decreased efficiency of fertilization. 2) By release in addition to nitrogen of NO and N₂O into the atmosphere, denitrification is involved in reactions that may cause destruction of the ozone layer. 3) Denitrification is the mechanism which balances dinitrogen fixation in the global N cycle. 4) Potential application of the process in the removal of nitrogen from waste materials with high nitrate concentrations.

Nitrate reduction is performed by many bacteria, fungi and by all plants assimilating nitrate as the nitrogen source. In this process, which occurs under both aerobic and anaerobic conditions, NO3- is reduced to NH₃. Contrary to assimilatory nitrate reduction, respiratory (dissmilatory) nitrate reduction is only known in bacteria. NO₃^- is reduced via NO₂^- and NO to N₂O and N₂. By using NO₃^-as an electron sink, the bacteria perform nitrate respiration which serves to generate energy as does O₂ respiration. Representative bacteria producing N₂ or N₂O are found in the genera Bacillus, Pseudomonas, Hyphomicrobium, Spirillum, Moraxella and Thiobacillus (T. denitrificans). The second route of anaerobic nitrate reduction leads to nitrite or ammonia. It occurs in a great number of genera, including Enterobacter, Escherichia, Bacillus, Micrococcus, Mycobacterium, Staphylococcus, Vibrio and Clostridium.

+4H +2H +2H +2H