Nitrogen fixation is a chemical process by which molecular nitrogen (N
2) in the air is converted into ammonia (NH
3) or related nitrogenous compounds in soil or aquatic systems. Atmospheric nitrogen is molecular dinitrogen, a relatively nonreactive molecule that is metabolically useless to all but a few microorganisms. Biological nitrogen fixation or diazotrophy is an important microbially mediated process that converts dinitrogen (N2) gas to ammonia (NH3) using the nitrogenase protein complex (Nif).
Nitrogen fixation is essential to life because fixed inorganic nitrogen compounds are required for the biosynthesis of all nitrogen-containing organic compounds, such as amino acids and proteins, nucleoside triphosphates and nucleic acids. As part of the nitrogen cycle, it is essential for agriculture and the manufacture of fertilizer. It is also, indirectly, relevant to the manufacture of all nitrogen chemical compounds, which includes some explosives, pharmaceuticals, and dyes.
Nitrogen fixation is carried out naturally in soil by microorganisms termed diazotrophs that include bacteria such as Azotobacter and archaea. Some nitrogen-fixing bacteria have symbiotic relationships with plant groups, especially legumes. Looser non-symbiotic relationships between diazotrophs and plants are often referred to as associative, as seen in nitrogen fixation on rice roots. Nitrogen fixation occurs between some termites and fungi. It occurs naturally in the air by means of NOx production by lightning.
All biological reactions involving the process of nitrogen fixation are catalysed by enzymes called nitrogenases. These enzymes contain iron, often with a second metal, usually molybdenum but sometimes vanadium.
Biological nitrogen fixation was discovered by Jean-Baptiste Boussingault in 1838. Later, in 1880, the process by which it happens was discovered by German agronomist Hermann Hellriegel and Hermann Wilfarth and was fully described by Dutch microbiologist Martinus Beijerinck.
"The protracted investigations of the relation of plants to the acquisition of nitrogen begun by Saussure, Ville, Lawes and Gilbert and others culminated in the discover of symbiotic fixation by Hellriegel and Wilfarth in 1887."
"Experiments by Bossingault in 1855 and Pugh, Gilbert & Lawes in 1887 had shown that nitrogen did not enter the plant directly. The discovery of the role of nitrogen fixing bacteria by Herman Hellriegel and Herman Wilfarth in 1886-8 would open a new era of soil science."
In 1901 Beijerinck showed that azotobacter chroococcum was able to fix atmospheric nitrogen. This was the first species of the azotobacter genus, so-named by him. It is also the first known diazotroph, the species that use diatomic nitrogen as a step in the complete nitrogen cycle.
The process is coupled to the hydrolysis of 16 equivalents of ATP and is accompanied by the co-formation of one equivalent of H
2. The conversion of N
2 into ammonia occurs at a metal cluster called FeMoco, an abbreviation for the iron-molybdenum cofactor. The mechanism proceeds via a series of protonation and reduction steps wherein the FeMoco active site hydrogenates the N
2 substrate. In free-living diazotrophs, nitrogenase-generated ammonia is assimilated into glutamate through the glutamine synthetase/glutamate synthase pathway. The microbial nif genes required for nitrogen fixation are widely distributed in diverse environments.
For example decomposing wood which has generally low content of nitrogen was shown to host diazotrophic community. Bacteria through fixation enrich wood substrate with nitrogen thus enabling deadwood decomposition by fungi.
Nitrogenases are rapidly degraded by oxygen. For this reason, many bacteria cease production of the enzyme in the presence of oxygen. Many nitrogen-fixing organisms exist only in anaerobic conditions, respiring to draw down oxygen levels, or binding the oxygen with a protein such as leghemoglobin.
Importance of nitrogen
Atmospheric nitrogen is inaccessible to most organisms. The nitrogen requirements for life is highly variable. Considering atom acquisition, for every 100 atoms of carbon, roughly 2 to 20 atoms of nitrogen are assimilated. The atomic ratio of Carbon(C): Nitrogen(N): Phosphorus(P) observed on average in planktonic biomass was originally described by Alfred Redfield. The Redfield Ratio, the stoichiometric relationship between C:N:P atoms, is 106:16:1.
The protein complex nitrogenase is responsible for catalyzing the reduction of nitrogen gas (N2) to ammonia (NH3). In Cyanobacteria, this enzyme system is housed in a specialize cell called the heterocyst. The production of the nitrogenase complex is genetically regulated, and the activity of the protein complex is dependent on ambient oxygen concentrations, and intra- and extracellular concentrations of ammonia and oxidized nitrogen species (nitrate and nitrite). Additionally, the combined concentrations of both ammonium and nitrate are thought to inhibit NFix, specifically when intracellular concentrations of 2-oxoglutarate (2-OG) exceed a critical threshold. The specialized heterocyst cell is necessary for the performance of nitrogenase as a result of its sensitivity to ambient oxygen.
Nitrogenase consist of two proteins, a catalytic iron-dependent protein, commonly referred to as MoFe protein and a reducing iron-only protein (Fe protein). There are three different iron dependent proteins, molybdenum-dependent, vanadium-dependent, and iron-only with all three nitrogenase proteins variations containing an iron protein component. Molybdenum-dependent nitrogenase is the most commonly present nitrogenase. The different types of nitrogenase can be determined by the specific iron protein component. Nitrogenase is highly conserved, gene expression through DNA sequencing can distinguish which protein complex is present in the microorganism and potentially being express. Most frequently, the nifH gene is used to identify the presence of molybdenum-dependent nitrogenase followed by closely related nitrogenase reductases (component II) vnfH and anfH representing vanadium-dependent and iron-only nitrogenase, respectively. In studying the ecology and evolution of nitrogen-fixing bacteria, the nifH gene is the biomarker most widely used. nifH has two similar genes anfH and vnfH that also encode for the nitrogenase reductase component of the nitrogenase complex
Diazotrophs are widespread within domain Bacteria including cyanobacteria (e.g. the highly significant Trichodesmium and Cyanothece), as well as green sulfur bacteria, Azotobacteraceae, rhizobia and Frankia. Several obligately anaerobic bacteria fix nitrogen including many (but not all) Clostridium spp. Some archaea also fix nitrogen, including several methanogenic taxa, which are significant contributors to nitrogen fixation in oxygen-deficient soils.
Cyanobacteria, commonly known as blue-green algae, inhabit nearly all illuminated environments on Earth and play key roles in the carbon and nitrogen cycle of the biosphere. In general, cyanobacteria can use various inorganic and organic sources of combined nitrogen, such as nitrate, nitrite, ammonium, urea, or some amino acids. Several cyanobacteria strains are also capable of diazotrophic growth, an ability that may have been present in their last common ancestor in the Archean eon. Nitrogen fixation not only naturally occurs in soils but also aquatic systems, including both freshwater and marine. Nitrogen fixation by cyanobacteria in coral reefs can fix twice as much nitrogen as on land—around 660 kg/ha/year. The colonial marine cyanobacterium Trichodesmium is thought to fix nitrogen on such a scale that it accounts for almost half of the nitrogen fixation in marine systems globally.
Species of nitrogen fixing cyanobacteria in fresh waters include: Aphanizomenon and Dolichospermum (previously Anabaena). Such species have specialized cells called heterocytes, in which nitrogen fixation occurs via the nitrogenase enzyme.
Root nodule symbioses
Plants that contribute to nitrogen fixation include those of the legume family—Fabaceae— with taxa such as kudzu, clover, soybean, alfalfa, lupin, peanut and rooibos. They contain symbiotic rhizobia bacteria within nodules in their root systems, producing nitrogen compounds that help the plant to grow and compete with other plants. When the plant dies, the fixed nitrogen is released, making it available to other plants; this helps to fertilize the soil. The great majority of legumes have this association, but a few genera (e.g., Styphnolobium) do not. In many traditional farming practices, fields are rotated through various types of crops, which usually include one consisting mainly or entirely of clover.
Fixation efficiency in soil is dependent on many factors, including the legume and air and soil conditions. For example, nitrogen fixation by red clover can range from 50 to 200 lb./acre.
Other nitrogen fixing families include:
- Parasponia, a tropical genus in the family Cannabaceae, which are able to interact with rhizobia and form nitrogen-fixing nodules
- Actinorhizal plants such as alder and bayberry can form nitrogen-fixing nodules, thanks to a symbiotic association with Frankia bacteria. These plants belong to 25 genera distributed across eight families.
The ability to fix nitrogen is present in other families that belong to the orders Cucurbitales, Fagales and Rosales, which together with the Fabales form a clade of eurosids. The ability to fix nitrogen is not universally present in these families. For example, of 122 Rosaceae genera, only four fix nitrogen. Fabales were the first lineage to branch off this nitrogen-fixing clade; thus, the ability to fix nitrogen may be plesiomorphic and subsequently lost in most descendants of the original nitrogen-fixing plant; however, it may be that the basic genetic and physiological requirements were present in an incipient state in the most recent common ancestors of all these plants, but only evolved to full function in some of them.
Betulaceae: Alnus (alders)
Several nitrogen-fixing symbiotic associations involve cyanobacteria (such as Nostoc):
Endosymbiosis in diatoms
Eukaryotic Nitrogenase Engineering
Some scientists are working towards introducing the genes responsible for nitrogen fixation directly into plant DNA. As all known examples of nitrogen fixation takes place in prokaryotes, transferring the functionality to eukaryotes such as plant is a challenge; one team is using yeast as their eukaryotic test organism. A major problem to overcome is the oxygen-sensitivity of the produced enzymes, as well as the energy requirements. Having the process taking place inside of mitocondria or chloroplasts is being considered.
The possibility that atmospheric nitrogen reacts with certain chemicals was first observed by Desfosses in 1828. He observed that mixtures of alkali metal oxides and carbon react at high temperatures with nitrogen. With the use of barium carbonate as starting material, the first commercial process became available in the 1860s, developed by Margueritte and Sourdeval. The resulting barium cyanide reacts with steam, yielding ammonia. A method for nitrogen fixation was first described by Henry Cavendish in 1784 using electric arcs reacting nitrogen and oxygen in air. This method was implemented in the Birkeland–Eyde process. The fixation of nitrogen by lightning is very similar natural occurring process.
In 1898 Frank and Caro developed a way to fix nitrogen in the form of calcium cyanamide. The Frank-Caro and Ostwald processes dominated industrial fixation until the discovery of the Haber process in 1909.
The most common ammonia production method is the Haber process. The Haber-Bosch nitrogen reduction process for industrial fertilizer production revolutionized modern day technology. Fertilizer production is now the largest source of human-produced fixed nitrogen in the terrestrial ecosystem. Ammonia is a required precursor to fertilizers, explosives, and other products. The Haber process requires high pressures (around 200 atm) and high temperatures (at least 400 °C), which are routine conditions for industrial catalysis. This process uses natural gas as a hydrogen source and air as a nitrogen source. The ammonia byproduct has resulted in an intensification of nitrogen fertilizer globally and is accredited with supporting the expansion of human population from roughly 2 billion in the early 20th century to roughly 7 billion people presently.
Much research has been conducted on the discovery of catalysts for nitrogen fixation, often with the goal of reducing energy requirements. However, such research has thus far failed to approach the efficiency and ease of the Haber process. Many compounds react with atmospheric nitrogen to give dinitrogen complexes. The first dinitrogen complex to be reported was Ru(NH 3)5(N2)2+.
Much research has been conducted on the discovery of catalysts for nitrogen fixation, often with the goal of reducing energy requirements. However, such research has thus far failed to approach the efficiency and ease of the Haber process. Many compounds react with atmospheric nitrogen to give dinitrogen complexes. The first dinitrogen complex to be reported was Ru(NH
2)2+. Some soluble complexes do catalyze nitrogen fixation.
Nitrogen can be fixed by lightning that converts nitrogen gas (N
2) and oxygen gas (O
2) present in the atmosphere into NO
x (nitrogen oxides). NO
x may react with water to make nitrous acid or nitric acid, which seeps into the soil, where it makes nitrate, which is of use to plants. Nitrogen in the atmosphere is highly stable and nonreactive due to the triple bond between atoms in the N
2 molecule. Lightning produces enough energy and heat to break this bond allowing nitrogen atoms to react with oxygen, forming NO
x. These compounds cannot be used by plants, but as this molecule cools, it reacts with oxygen to form NO
2. This molecule in turn reacts with water to produce HNO
3 (nitric acid), or its ion NO−
3 (nitrate), which is usable by plants.
- Birkeland–Eyde process: an industrial fertiliser production process
- George Washington Carver: an American botanist
- Denitrification: an organic process of nitrogen release
- Nitrification: biological production of nitrogen
- Nitrogen cycle: the flow and transformation of nitrogen through the environment
- Nitrogen deficiency
- Nitrogen fixation package for quantitative measurement of nitrogen fixation by plants
- Nitrogenase: enzymes used by organisms to fix nitrogen
- Ostwald process: a chemical process for making nitric acid (HNO
- Push–pull technology: the use of both repellent and attractive organisms in agriculture
- Carbon fixation
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