Essay on the Nitrogen Cycle

After reading this essay you will learn about the nitrogen cycle.

Nitrogen is the most important structural element of all living organisms. There is a vast store of nitrogen in the air, but animals and the majority of plants are unable to fix atmospheric nitrogen. The nitrogen cycle is chiefly concerned with the incorporation of atmospheric gaseous nitrogen and organic nitrogen of dead plants and animals into the forms that are usable by higher plants.

The scarcity of suitable nitrogenous compounds is a major problem in the maintenance of soil fertility. For this reason the transformation of nitrogen has attracted considerable attention from soil microbiologists. Higher plants generally require nitrogen in the form of nitrates, although ammonia and some organic nitrogenous compounds are also utilized to a lesser extent.

The overall transformations in which micro-organisms are involved range from elementary nitrogen to protein and other complex organic nitrogenous compounds, with a large number of intermediary substances. The distinctive processes of nitrogen cycle are ammonification of cellular nitrogen, nitrification, nitrate reduction, de-nitrification and nitrogen fixation.

1. Ammonification:

Proteins and other organic nitrogenous compounds of living and dead organisms, which find their into the soil, are decomposed by micro-organisms. These compounds are hydrolyzed by various proteolytic enzymes to amino acids and similar compounds. The amino groups (-NH₂) are split off to form ammonia (NH₃).

Release of ammonia from organic nitrogenous compounds is termed ammonification. Proteolytic enzymes are elaborated by some Clostridia, many fungi, and actinomycetes and to a lesser degree by Pseudomonas, Bacillus, and Proteus species. The ultimate products of proteolysis are amino acids.

Amino acids are deaminate under aerobic conditions (oxidative deamination), with the liberation of ammonia. However, if protein decomposition occurs under anaerobic condition, amino acids are converted to amines and related compound [putrefaction). These reactions are most commonly brought about by Clostridium species.

Eventually amines are oxidized in the presence of air with liberation of ammonia. Similarly urea present in the urine of man and animals is also decomposed with liberation of ammonia by several micro-organisms specially, by Micrococcus species Proteus species etc.

Ammonium ion toxic even at low ion concentration and is therefore never allowed to accumulate. Most of it is assimilated by soil micro-organisms and some of it is utilized by plants as a source of nitrogen. Under favourable conditions it is oxidized first to nitrite and then to nitrate by specific groups of organisms.

2. Nitrification:

The oxidation of ammonia to nitrate is called nitrification. The process consists of two steps and is carried out by two specific groups of organisms of the family Nitro-bacteriaceae. These are strict autotrophs and obtain their energy from the oxidation of ammonia to nitrites and nitrites to nitrates. The first step, the oxidation of ammonia to nitrite, is called nitrosification.

2NH₃ + 3O₂→ 2HNO₂ + 2H₂O + 70,000 cals

This is accomplished by bacteria of the genera Nitrosomonas, Nitrosococcus, Nitrosospira, and Nitrosocystis. The nitrites formed by these organisms are toxic to plants as well as to the organisms forming it. Fortunately, nitrites are removed by further oxidation to nitrates by bacteria of the genus Nitrobacter.

2HNO₂ + O₂→2 HNO₃ + 20,000 cals.

Nitrification proceeds very rapidly in well-aerated manure piles. The nitrates from such sources were used for the manufacture of gun-powder during the Napoleonic wars, long before the microbial process of nitrification was known. Nitrates are readily used by plants and many micro-organisms.

Certain heterotrophic bacteria (e.g. Streptomyces and Nocardia) oxidize ammonia to nitrite. However, several species of fungi (e.g. Penicillium, Aspergillus, Cephalosporium) oxidize both ammonia to nitrite and nitrite to nitrate.

3. Nitrate Reduction:

The nitrification process is reversed by many micro-organisms, which are capable of reducing nitrate to nitrite and then to ammonia. This process is called nitrate reduction and involves several reactions.

The overall result is:

HNO₃ + 4H₂→ NH₃ + 3H₂O

Since organisms are able to obtain cellular nitrogen through ammonia assimilation the process is called assimilatory nitrate reduction. A large number of microbial species including bacteria, yeast, and filamentous fungi assimilate nitrate nitrogen through this process.

4. De-Nitrification:

Certain micro-organisms are capable of reducing nitrates to nitrites, and subsequently to gaseous nitrogen (e.g. nitrous oxide or free nitrogen). This process is called de-nitrification. Under anaerobic conditions and in presence of an abundant supply of organic compounds, which serve as hydrogen donors, nitrate serves as an electron acceptor.

NO₃^– + 2e^– + 2H⁺→ NO₂^– + H₂O

Nitrite is ultimately reduced to molecular nitrogen through several reactions which are not clearly understood. The overall reaction is:

2 NO₃^– + 10e^– + 12H⁺→ N₂ + 6 H₂O

When nitrate is used as a source of electron acceptors, there is a net loss of nitrogen from the soil. This process is, therefore, called dissimilatory nitrate reduction. Nitrogen loss by de-nitrification occurs during seasonal flooding of the land, or as a result of over-irrigation of poorly drained land.

Lack of fertility of constantly wet soils is due to the growth of nitrate reducing anaerobic species. Some of these organisms are Thiobacillus denitrificans, Micrococcus denitrificans, various species of Clostridium and some species of Serratia, Pseudomonas and Achromobacter.

5. Nitrogen Fixation:

The nitrogen cycle show obvious loss of fixed nitrogen, particularly through de-nitrification. Secondly there is a loss into the sea which is not recovered. Thirdly there is a loss by explosives, while this may seem minor except in war, it is actually reasonably large because of blasting of rock, earth etc.

These are relatively large losses and eventually the fixed nitrogen would be used up. On the other hand animals and plants are unable to utilize in any way vast stores of nitrogen in the air. A small amount of non-biological nitrogen fixation occurs in the atmosphere through lightning discharges. Nitric oxide which is formed reacts with water to produce nitric acid.

This combines with ammonia and some ammonium nitrate is brought to the soil. There are a few man-made processes (Haber, Berkland-Eyde, Cynamide) where nitrogen is fixed, but they require rather drastic conditions of temperature and pressure. In contrast to this micro-organisms fix atmospheric nitrogen very smoothly at 1 atmospheric pressure and at a temperature of 20°C.

There are two main groups of nitrogen-fixing organisms according to their mode of fixation:

(a) Symbiotic nitrogen fixers: those capable of fixing molecular nitrogen by living in the roots of leguminous plants; and

(b) Non-symbiotic nitrogen fixers: those capable of fixing molecular nitrogen to cellular nitrogen independently of other living organisms.

a. Symbiotic Nitrogen Fixation:

It has been widely recognised for centuries that most crops decrease the fertility of the soil, but leguminous crops increase it. Legumes thus restore or renew the soil. This is because legumes fix nitrogen from the air. This was first demonstrated by the French chemist Boussingault in 1837.

He observed that nitrogen present in the leguminous plants exceeded the nitrogen present in the seeds and the soil. Hellriegel and Willfarth in Germany and Atwater in USA conclusively proved that nitrogen is fixed by certain bacteria living in root nodules of the leguminous plants.

Since neither the plant nor the bacterium can fix atmospheric nitrogen independently, the process, therefore, is called symbiotic nitrogen fixation.

The root nodule bacteria were later isolated by Beijerinck is 1888 and were classified and grouped in the genus Rhizobium (Rhixo means root in Greek). These are gram negative, motile, aerobic, non-spore forming bacteria. They are mainly rod shaped, but when isolated from nodules a variety of morphological shapes are observed.

Rhizobium:

Rhizobium species invade the root hairs of leguminous plants. The bacteria aggregate as threads and penetrate the plant cells. The presence of bacteria stimulates the multiplication of infected cells, resulting in the formation of nodules. The legume, the bacteria, and the nodule constitute the system for the symbiotic nitrogen fixation, where both the bacteria and the plant benefit by the association.

The bacteria obtain their nutrients and source of energy from the plant, and in turn, fix atmospheric nitrogen and made it available to the plant. Bacteria isolated from root nodules of one legume are not necessarily capable of producing nodules on another legume.

A certain degree of specificity therefore exists between the bacteria and legumes. A number of bacterial species are named on this basis of susceptible plants. Generally eight groups have been recognised.

Rhizobium species of proper type which will form a symbiotic association with a particular legume may be absent in the soil. Legume seeds before planting are, therefore, inoculated with strains of Rhizobium of known effectiveness.

This is called pre-inoculation. Bacterial cultures for this purpose are available commercially. Seeds inoculated with such cultures assure the presence of desirable strains of Rhizobia as soon as young rootlets are formed.

b. Non-Symbiotic Nitrogen Fixation:

Winogradsky in Russia and Beijerinck in Holland independently tried to isolate micro-organisms which can fix atmospheric nitrogen without symbiotic aid. Winogradsky was the first to succeed and isolated gram positive, spore forming, and anaerobic bacillus known as Clostridum pasteurianum.

Beijerinck isolated gram negative, non-spore forming, aerobic, pleomorphic coccoid or rod-shaped bacteria belonging, to the genus Azotobacter (Azote means nitrogen in French). However, in recent years a large number of organisms have been isolated that fix nitrogen non-symbiotically. This has been achieved by cultivating the micro-organisms in presence of nitrogen labelled with isotopic ¹⁵N.

Mechanism of Nitrogen Fixation:

The mechanism of nitrogen fixation is not fully understood, but the first product detectable by isolopic studies is ammonia. Reduction of nitrogen to ammonia requires a valence change (0 to -3). Six electrons are therefore required to reduce one mole of nitrogen to two moles of ammonia.

N₂ + 6H⁺ + 6e^– → 2NH₃

This requires the breakup of nitrogen bonds (N = N). It is agreed that atoms of oxygen are separated in biological oxidation through change in valency of the metal ion (Fe) in the enzyme cytochrome oxidase. It is postulated that atoms of nitrogen are separated trough change in the valency of the metal ion (molybdenum) bound to the enzyme involved in the reduction of nitrogen.

Non-symbiotic nitrogen fixation is better understood because a cell free nitrogen fixing system is available. A cell free preparation, made of two fractions, has been isolated from CI. pasteurianum. Fraction one is known as electron donating and ATP generating system. It is involved in the metabolism of pyruvic acid.

CH₃COCOOH + Pi → CH₃COP + CO₂ + 2H⁺

CH₃COP + ADP → CH₃COOH + ATP

The second fraction involved in actual nitrogen fixation includes the enzyme nitrogenase. It is repressed by ammonium ions. It requires ferredoxin (non-haem Fe compound) or flavodoxin (FMN containing protein) as a carrier of reducing power. Ferredoxin or flavodoxin (Fd) is the first reducer, which in turn reduces nitrogen bound to the enzyme nitrogenase in presence of ATP.

Fd (oxidized) + nH → Fd (reduced)

Fd (red) + Nitrogenase + Nitrogen → Fd (oxd) + N₂ – Nitrogenase (red)

N₂ – Nitrogenase (red) + nATP → NH₃ + Nitrogenase + n ADP + nPi

Inorganic intermediates between N₂ and NH3 have not been detected. It is therefore presumed that reduction takes place directly on the enzyme surface (Fig. 18.40)

The enzyme nitrogenase has been studied and following characteristics have been noted:

(1) The enzyme is made up of at least two components; one contains both Fe and Mo, and the other contains only Fe.

(2) The enzyme can reduce other substrates besides nitrogen, such as nitrous oxide, azide, cyanide, acetylene etc.

(3) If no suitable electron acceptor is available, the enzyme forms molecular hydrogen

2H⁺ + 2e^– → H₂