In this article we will discuss about the Nitrogen Cycle.
Nitrogen Cycle:
Nitrogen is the fourth most prevalent element in living systems. It is a constituent of a number of organic compounds like amino acids, proteins, nucleotides, nucleic acid, hormones, chlorophyll, many vitamins, etc.
However, its availability from soil is limited and even for that plants have to compete with microbes both in natural and agricultural ecosystems. Nitrogen is available in the atmosphere in abundance (78% of atmosphere as di-nitrogen or N2) but plants cannot directly absorb the same.
Therefore, nitrogen is the most critical element. A regular supply of nitrogen to the plants is maintained through nitrogen cycle. Nitrogen cycle is regular circulation of nitrogen amongst living organisms, reservoir pool in the atmosphere and cycling pool in the lithosphere. Nitrogen compounds are obtained from reservoir pool through nitrogen fixation.
Reservoir pool is replenished through de-nitrification of nitrates and release of nitrogen from decaying organic matter. Cycling pool is augmented by ammonification and nitrification. Plants obtain nitrogen from soil as NO3– (nitrate), NH4+ (ammonium) and NO2– (nitrite) ions. Nitrate and nitrite are reduced to ammonium state which is then incorporated into amino acids, proteins and other organic substances.
Nitrogen Fixation:
It is the conversion of inert atmospheric nitrogen or di-nitrogen (N2) into utilizable compounds of nitrogen like nitrate, ammonia, amino acids, etc. There are two methods of nitrogen fixation— abiological and biological. Abiological nitrogen fixation is further of two kinds, natural and industrial.
Natural Abiological Nitrogen Fixation:
Atmospheric nitrogen combines with oxygen in the presence of electric discharges, ozonization and combustion. Different types of nitrogen oxides are produced. The nitrogen oxides dissolve in water and give rise to hyponitrous, nitrous and nitric acids. They enter soil along with rain water forming hyponitrites, nitrites and nitrates.
H2O + 2NO → HNO + HNO2
H2O + 2NO2 → HNO2 + HNO3
H2O + N2O5→ 2HNO3
Industrial Abiological Nitrogen Fixation:
Ammonia is produced industrially by direct combination of nitrogen with hydrogen (got from water) at high temperature and pressure. It is changed to various types of fertilizers including urea.
Biological Nitrogen Fixation:
It is the second most important natural process and the major source of nitrogen fixation which is performed by two types of prokaryotes, bacteria and cyanobacteria (= blue green algae).
They include both free living and symbiotic forms:
(a) Free Living Nitrogen Fixing Bacteria:
Azotobacter, Beijerinckia (both aerobic) and Bacillus, Klebsiella, Clostridium (all anaerobic) are saprotrophic bacteria that perform nitrogen fixation. Desulphovibrio is chemotrophic nitrogen fixing bacterium. Rhodopseudomonas, Rhodospirillum and Chromatium are nitrogen fixing anaerobic photoautotrophic bacteria. Free living nitrogen fixing bacteria add 10-25 kg of nitrogen/ha/annum.
(b) Free Living Nitrogen Fixing Cyanobacteria:
Many free living blue- green algae (BGA) or cyanobacteria perform nitrogen fixation, e.g., Anabaena, Nostoc, Calothrix, Lyngbia, Aulosira, Cylindrospermum, Trichodesmium. They add 20-30 kg of nitrogen per hectare of soil and water bodies.
Cyanobacteria are also important ecologically as they occur in waterlogged soils where denitrifying bacteria can be active. Aulosira fertilissima is the most active nitrogen fixer in Rice fields while Cylindrospermum is active in Sugarcane and Maize fields.
(c) Symbiotic Nitrogen Fixing Cyanobacteria:
Anabaena and Nostoc species are common symbionts in lichens, Anthoceros, Azolla and Cycad roots. Azolla pinnata (a water fern) has Anabaena azollae in its fronds. It is often inoculated to Rice fields for nitrogen fixation.
(d) Symbiotic Nitrogen Fixing Bacteria:
Rhizobium is nitrogen fixing bacterial symbiont of papilionaceous roots. Sesbania rostrata has Rhizobium in root nodules and Aerorhizobium in stem nodules.
Frankia is symbiont in root nodules of several nonlegume plants like Casuarina (Australian Pine), Myrica and Alnus (Alder). Xanthomonas and Mycobacterium form symbiotic association with the leaves of several members of rubiaceae and myrsinaceae (e.g., Ardisia).
Both Rhizobium and Frankia live free as aerobes in the soil but are unable to fix nitrogen. They develop the ability to fix nitrogen only as a symbiont when they become anaerobic. Rhizobium is rod-shaped bacterium while Frankia is an actinomycete.
Out of these Rhizobium is the most important for crop lands because it is associated with pulses and other legumes of family fabaceae, e.g., Chick Pea or Gram (Cicer arietinum), Pigeon Pea or Red Gram (Cajanus cajan), Garden or Edible Pea (Pisum sativum), Soya bean (Glycine max), Lentil (Lens culinaris), Green Gram (Vigna radiata = Phaseolus aureus), Black Gram (Vigna or Phaseolus mungo), Sweet Clover, Sweet Pea, Alfalfa, Broad Bean, Clover Bean. Several species of the bacterium (e.g., Rhizobium leguminosarum, R. meliloti) live in the soil.
They are unable to fix nitrogen by themselves. Roots of a legume secrete chemical attractants (flavonoids and betaines). Bacteria collect over the root hairs, release nod factors that cause curling of root hairs around the bacteria, degradation of cell wall and formation of an infection thread enclosing the bacteria (Fig. 12.11).
Infection thread grows along with multiplication of bacteria. It branches and its ends come to lie opposite protoxylem points of vascular strand. The infected cortical cells dedifferentiate and start dividing. It produces swellings or nodules.
Nodule formation is stimulated by auxin produced by cortical cells and cytokinin liberated by invading bacteria. The infected cells enlarge. Bacteria stop dividing and form irregular polyhedral structures called bacteriods (Fig. 12.12). However, some bacteria retain normal structure, divide and invade new areas. In an infected cell bacteriods occur in groups surrounded by host membrane.
The host cell develops a pinkish pigment called leg-haemoglobin (Lb). It is oxygen scavenger and is related to blood pigment haemoglobin. It protects nitrogen fixing enzyme nitrogenase from oxygen. Symbiotic nitrogen fixation requires cooperation of Nod genes of legume, nod, nif and fix gene clusters of bacteria.
Mechanism of Nitrogen Fixation:
Nitrogen fixation requires (i) a reducing power like NADPH, FMNH2 (ii) a source of energy like ATP (iii) enzyme di-nitrogenase and (iv) compounds for trapping ammonia formed by the reduction of di-nitrogen. Enzyme nitrogenase has iron and molybdenum. Both of them take part in attachment of a molecule of nitrogen (N2).
Bonds between the two atoms of nitrogen become weakened by their attachment to the metallic components. The weakened molecule of nitrogen is acted upon by hydrogen (Fig. 12.13) from a reduced coenzyme. It produces dimide (N2H2), hydrazine (N2H4) and then ammonia (2NH3).
Ammonia is not liberated. It is toxic in even small quantities. The nitrogen fixers protect themselves from it by providing organic acids. The reaction between ammonia and organic acids gives rise to amino acids.
N5 + 8e– 8H++16ATP- di-nitrogenase → 2NH3 + 2H++ 16ADP + l6Pi
Ammonia + α-ketoglutarate + NAD(P)H- dehydrogenase → Glutamate + NAD(P)+ + H2O
Symbiotic nitrogen fixing organisms hand over a part of their fixed nitrogen to the host in return for shelter and food. Free living nitrogen fixers do not immediately enrich the soil. It is only after their death that the fixed nitrogen enters the cycling pool. It occurs in two steps, ammonification and nitrification.
Ammonification:
It is carried out by decay causing organisms. They act upon nitrogenous excretions and proteins of dead bodies of living organisms, e.g., Bacillus ramosus, B. vulgaris, B. mesentericus, Actinomyces. Proteins are first broken up into amino acids. The latter are deaminated. Organic acids released in the process are used by microorganisms for their own metabolism.
Ammonia does not remain in the gaseous state in the soil but is changed to ionic form (NH+). It can be used by plants directly provided pH of soil is more than 6 and the plant contains abundant organic acids. Unlike nitrates, very few plants can store ammonium ions (e.g., Begonia, Oxalis).
Nitrification:
It is the phenomenon of conversion of ammonium nitrogen to nitrate nitrogen. It is performed in two steps— nitrite formation and nitrate formation. Both the steps can be carried out by Aspergillus flavus. In the first step, ammonium ions are oxidised to nitrites Nitrosococcus, Nitrosomonas. Nitrites are changed to nitrates in the second step, e.g., Nitrocystis, Nitrobacter.
Most of the bacteria performing nitrification (e.g., Nitrosococcus, Nitrosomonas, Nitrobacter) are chemoautotrophs. They use the energy liberated during nitrification in synthesis of organic substances from CO2 and a hydrogen donor. They are thus autotrophs which do not use solar energy for synthesis of food.
De-nitrification:
Under anaerobic conditions (e.g., water logging, oxygen depletion), some microorganisms use nitrate and other oxidised ions as source of oxygen. In the process, nitrates are reduced to gaseous compounds of nitrogen. The latter escape from the soil. Common bacteria causing de-nitrification of soil are Pseudomonas denitrificans, Thiobacillus denitrificans, Micrococcus denitrificans.
Nitrogen oxides escaping into atmosphere or formed during abiological fixation can also be broken down by raidations to form molecular nitrogen. De-nitrification of soil not only depletes the soil of an important nutrient but also causes acidification which is equally harmful in solubilisation of harmful metals.
Nitrate Assimilation:
Nitrate is the most important source of nitrogen to the plants. It can accumulate in the cell sap of several plants and take part in producing osmotic potential. However it cannot be used as such by the plants. It is first reduced to level of ammonia before being incorporated into organic compounds. Reduction of nitrate occurs in two steps.
(i) Reduction of Nitrate to Nitrite:
It is carried out by the agency of an inducible enzyme called nitrate reductase. The enzyme is a molybdoflavoprotein. It requires a reduced coenzyme (NADH or NADPH) for its activity. The reduced coenzyme is brought in contact with nitrate by FAD or FMN.
(ii) Reduction of Nitrite:
It is performed by enzyme nitrite reductase. The enzyme is a metalloflavoprotein which contains copper and iron. It occurs inside chloroplasts in the leaf cells and leucoplasts of other cells. In contrast nitrate reductase is found attached loosely to cell membrane. Nitrite reductase requires reducing power.
It is NADPH in illuminated cells and NADH in others. The process of reduction also requires ferredoxin which occurs in higher plants mostly in green tissues. Therefore, it is presumed that in higher plants either nitrite is trans-located to leaf cells or some other electron donor (like FAD) operates in un-illuminated cells. The product of nitrite reduction is ammonia.
Ammonia is not liberated. It combines with some organic acids to produce amino acids. Amino acids then form various types of nitrogenous compounds.
Synthesis of Amino:
The first organic compounds of nitrogen assimilation are ammo acids.
They are synthesised by following three methods:
1. Reductive Amination:
In the presence of dehydrogenase (e.g., Glutamate dehydrogenase, Aspartate dehydrogenase), a reduced coenzyme (NADH or NADPH), ammonia can directly combine with a keto organic acid like a-ketoglutaric acid and oxaloacetic acid to form amino acid.
2. Catalytic Amidation:
Ammonia combines with catalytic amounts of glutamic acid in the presence of ATP and enzyme glutamine synthetase. It produces an amide called glutamine. Glutamine reacts with a-ketoglutaric acid in the presence of enzyme glutamate synthetase to form two molecules of glutamate. Reduced co-enzyme (NADH or NADPH) is required.
3. Transamination:
It is transfer of amino group (> CH NH2) of one amino acid with the keto group (> С = О) of keto acid. The enzyme required is transaminase or aminotransferase. Glutamic acid is the primary amino acid involved in transfer of amino group (to as many as seventeen amino acids).
Amides:
They are amino acid derivatives in which – OH component of carboxylic group (- COOH) is replaced by another amino group (- NH2). Amides are, therefore, double aminated keto acids. The two most common amides are glutamine and asparagine.
They are formed by amidation of glutamic acid and aspartic acid respectively. Another common amide is vitamin niacin amide (niacin a). Glutamine and asparagine are components of proteins along with amino acids.
Their formation requires ATP, ammonia and synthetase enzyme (glutamine synthetase, asparagine synthetase). Amides perform two other functions— storage of excess nitrogen and transport.
Transport generally occurs via xylem. Ureides (degraded urea) are employed as a means of nitrogen transport from the nodules of some legumes (e.g., Soya-bean) and re-transport from the ageing organs. They have a high nitrogen to carbon ratio.