Nitrogen fixation

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Nitrogen fixation is the natural process, either biological or abiotic, by which nitrogen (N2) in the atmosphere is converted into ammonia (NH3). This process is essential for life because fixed nitrogen is required to biosynthesize the basic building blocks of life, e.g., nucleotides for DNA and RNA and amino acids for proteins. Nitrogen fixation also refers to other biological conversions of nitrogen, such as its conversion to nitrogen dioxide.

Nitrogen fixation is utilized by numerous prokaryotes, including bacteria, actinobacteria, and certain types of anaerobic bacteria. Microorganisms that fix nitrogen are called diazotrophs. Some higher plants, and some animals (termites), have formed associations (symbioses) with diazotrophs. Nitrogen fixation also occurs as a result of non-biological processes. These include lightning, industrially through the Haber-Bosch Process, and combustion. Biological nitrogen fixation was discovered by the Dutch microbiologist Martinus Beijerinck.

Tu et al. (2009) grew peanut (Arachis hypogaea L.) plants from seed to maturity outdoors near Raleigh, North Carolina (USA) in open-top chambers under adequately watered and fertilized conditions, while exposing the plants to (1) charcoal-filtered air, which was thus ozone-free, (2) ambient air of unaltered ozone (O3) concentration, and (3) air containing 1.6 times the ambient O3 concentration. All of these O3 treatments were exposed to air of 376, 550, and 730 ppm CO2, while the researchers made many plant physiological measurements. At the end of the period they harvested the crop and measured its final steam, leaf, and pod biomass. The four researchers found “at mid-vegetative growth, elevated CO2 significantly reduced leaf nitrogen concentrations by up to 44%,” but “plant nitrogen concentrations only differed by 8% among CO2 treatments at harvest while N2 fixation was increased.” They state their findings suggest “symbiotic N2 fixation is important for maintaining seed N concentrations and that CO2 enhancement of symbiotic N2 fixation may compensate for low soil N availability.”

One year later, Prevost et al. (2010) grew soybean (Glycine max [L.] Merr. cv. Lotus) plants from seed in 24-cm-deep pots filled with a sandy loam soil that was watered and fertilized according to standard agricultural procedures for a period of six weeks within controlled-environment chambers maintained at atmospheric CO2 concentrations of either 400 or 800 ppm, after inoculating either the soil or the seeds with either a reference strain (532c) of the nitrogen-fixing bacteria Bradyrhizobium japonicum, which is widely used in commercial operations in Canada, or with one of two strains of B. japonicum that are indigenous to the soils of Quebec (5Sc2 or 12NS14). They found “elevated CO2 increased mass (+63%) and number (+50%) of soybean nodules, particularly medium and large, allowed a deeper nodule development, and increased shoot dry weight (+30%), shoot carbon uptake (+33%) and shoot nitrogen uptake (+78%), compared to ambient CO2.”

The four Canadian scientists state their results “constitute the first report showing that elevated CO2 affects nodule size by allowing a greater production of large nodules, and influences nodule localization by favoring deeper nodule development on roots.” Regarding the significance of these findings, they write, “medium and/or large nodules may confer advantages to legumes,” since “they have been shown to improve drought tolerance of soybean (King and Purcell, 2001) and to exhibit higher nitrogenase activity in peanut (Tajima et al., 2007).” Also, they say their finding that “both shoot nitrogen and carbon uptakes are stimulated by elevated CO2 agrees with Rogers et al. (2009), who stated that photosynthetic activity in legumes under elevated CO2 does not acclimate [decrease with time] under optimal growing conditions, since the additional photosynthates produced are allocated to root nodules for N2 fixation.” And they note similar increases in nodule mass and number have been observed “with other legume species (Schortemeyer et al., 2002; Cabrerizo et al., 2001; Haase et al., 2007),” as well as “with soybean under drought (Serraj et al., 1998).” These findings bode well for legume farmers of the future and for the people and livestock that will consume their produce.


Biological nitrogen fixation

Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by an enzyme called nitrogenase. The formula for BNF is:

N2 + 8 H+ + 6 e− → 2 NH3 + H2

The process is coupled to the hydrolysis of 16 equivalents of ATP and is accompanied by the co-formation of one molecule of H2. In free-living diazotrophs, the nitrogenase-generated ammonium is assimilated into glutamate through the glutamine synthetase/glutamate synthase pathway.

Enzymes responsible for nitrogenase action are very susceptible to destruction by oxygen. (In fact, 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.

Plants that contribute to nitrogen fixation include the legume family – Fabaceae – with taxa such as clover, soybeans, alfalfa, lupines, peanuts, and rooibos. They contain symbiotic bacteria called Rhizobia 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 and 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 and organic farming practices, fields are rotated through various types of crops, which usually includes one consisting mainly or entirely of clover or buckwheat (family Polygonaceae), which were often referred to as "green manure."

Although by far the majority of nitrogen-fixing plants are in the legume family Fabaceae, there are a few non-leguminous plants, such as alder and black locust, that can also fix nitrogen. These plants, referred to as "actinorhizal plants", consist of 24[verification needed] genera of woody shrubs or trees distributed among in 8 plant families. The ability to fix nitrogen is not universally present in these families. For instance, of 122 genera in the Rosaceae, only 4 genera are capable of fixing nitrogen. All these families belong to the orders Cucurbitales, Fagales, and Rosales, which together with the Fabales form a clade of eurosids. In this clade, Fabales were the first lineage to branch off; 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 last common ancestors of all these plants, but only evolved to full function in some of them:

Family: Genera

Betulaceae: Alnus (alders)

Cannabaceae: Trema







Coriariaceae: Coriaria

Datiscaceae: Datisca


Elaeagnus (silverberries)

Hippophae (sea-buckthorns)

Shepherdia (buffaloberries)




Morella arborea













Cercocarpus (mountain mahoganies)

Chamaebatia (mountain miseries)


Purshia/Cowania (bitterbrushes/cliffroses)

There are also several nitrogen-fixing symbiotic associations that involve cyanobacteria (such as Nostoc):

Some lichens such as Lobaria and Peltigera

Mosquito fern (Azolla species)



Microorganisms that fix nitrogen (Diazotrophs)

Cyanobacteria Azotobacteraceae Rhizobia Frankia

Nitrogen fixation by rhizobia

Rhizobia are soil, Gram-negative bacteria with the unique ability to establish a N2-fixing symbiosis on legume roots and on the stems of some aquatic legumes. During this interaction bacteroids, as rhizobia are called in the symbiotic state, are contained in intracellular compartments within a specialized organ, the nodule, where they fix N2.

Nitrogen fixation by cyanobacteria

Cyanobacteria inhabit nearly all illuminated environments on Earth and play key roles in the carbon and nitrogen cycle of the biosphere. In general, cyanobacteria are able to utilize a variety of inorganic and organic sources of combined nitrogen, like nitrate, nitrite, ammonium, urea, or some amino acids. Several cyanobacterial strains are also capable of diazotrophic growth. Genome sequencing has provided a large amount of information on the genetic basis of nitrogen metabolism and its control in different cyanobacteria. Comparative genomics, together with functional studies, has led to a significant advance in this field over the past years. 2-Oxoglutarate has turned out to be the central signalling molecule reflecting the carbon/nitrogen balance of cyanobacteria. Central players of nitrogen control are the global transcriptional factor NtcA, which controls the expression of many genes involved in nitrogen metabolism, as well as the PII signalling protein, which fine-tunes cellular activities in response to changing C/N conditions. These two proteins are sensors of the cellular 2-oxoglutarate level and have been conserved in all cyanobacteria. In contrast, the adaptation to nitrogen starvation involves heterogeneous responses in different strains. Nitrogen fixation by cyanobacteria in coral reefs can fix twice the amount of nitrogen than on land–around 1.8 kg of nitrogen is fixed per hectare per day.

Chemical nitrogen fixation

Haber process

Nitrogen can also be artificially fixed as ammonia for use in fertilizers, explosives, or in other products. The most common method is the Haber process. Artificial fertilizer production is now the largest source of human-produced fixed nitrogen in the Earth's ecosystem.

The Haber process requires high pressures (around 200 atm) and high temperatures (at least 400 °C), routine conditions for industrial catalysis. This highly efficient process uses natural gas as a hydrogen source and air as a nitrogen source.

Dinitrogen complexes

Dinitrogen complex

Much research has been conducted on the discovery of catalysts for nitrogen fixation, often with the goal of reducing the energy required for this conversion. However, such research has thus far failed to even approach the efficiency and ease of the Haber process. Many compounds react with atmospheric nitrogen under ambient conditions. For example, lithium metal converts to lithium nitride under an atmosphere of nitrogen. Treatment of the resulting nitride gives ammonia.

The first dinitrogen complex was reported in 1965 based on ammonia coordinated to ruthenium ([Ru(NH3)5(N2)]2+). Research in chemical fixation from then on focused on transition metal complexes. Since then, a large number of transition metal compounds that contain dinitrogen as a ligand have been discovered. The dinitrogen ligand can either be bound to a single metal or bridge two (or more) metals. The coordination chemistry of dinitrogen is complex and currently under intense investigation. This research may lead to new ways of using dinitrogen in synthesis and on an industrial scale.

Ambient nitrogen reduction

Catalytic chemical nitrogen fixation at temperatures considerably lower than the Haber process is an ongoing scientific endeavor. Nitrogen was successfully converted to ammonia and hydrazine by Alexander E. Shilov in 1970 The first example of homolytic cleavage of dinitrogen under mild conditions was published in 1995. Two equivalents of a molybdenum complex reacted with one equivalent of dinitrogen, creating a triple bonded MoN complex. Since then, this triple bounded complex has been used to make nitriles.

The first catalytic system converting nitrogen to ammonia at room temperature and pressure was discovered in 2003 and is based on another molybdenum compound, a proton source, and a strong reducing agent. However, this catalytic reduction fixates only a few nitrogen molecules.

In 2011 Arashiba et al. reported another system with a catalyst again based on molybdenum but with a diphosphorus pincer ligand


Cabrerizo, P.M., Gonzalez, E.M., Aparico-Tejo, P.M., and Aresse-Igor, C. 2001. Continuous CO2 enrichment leads to increased nodule biomass, carbon availability to nodules and activity of carbon-metabolizing enzymes but does not enhance specific nitrogen fixation in pea. Physiologia Plantarum 113: 33–40.

Haase, S., Neumann, G., Kania, A., Kuzyakov, Y., Romheld, V., and Kandeler, E. 2007.

Elevation of atmospheric CO2 and N-nutritional status modify nodulation, nodule-carbon supply, and root exudation of Phaseolus vulgaris L. Soil Biology & Biochemistry 39: 2208–2221.

King, C.A. and Purcell, L.C. 2001. Soybean nodule size and relationship to nitrogen fixation response to water deficit. Crop Science 41: 1099–1107.

Prevost, D., Bertrand, A., Juge, C., and Chalifour, F.P. 2010. Elevated CO2 induces differences in nodulation of soybean depending on bradyrhizobial strain and method of inoculation. Plant and Soil 331: 115–127.

Rogers, A., Ainsworth, E.A., and Leakey, A.D.B. 2009. Will elevated carbon dioxide concentration amplify the benefits of nitrogen fixation in legumes? Plant Physiology 151: 1009–1016.

Shortemeyer, M., Atkin, O.K., McFarlane, N., and Evans, J.R. 2002. N2 fixation by Acacia species increases under elevated atmospheric CO2. Plant, Cell and Environment 25: 567–579.

Serraj, R., Sinclair, T.R., and Allen, L.H. 1998. Soybean nodulation and N2 fixation response to drought under carbon dioxide enrichment. Plant, Cell and Environment 21: 491–500.

Tajima, R., Lee, O.N., Abe, J., Lux, A., and Morita, S. 2007. Nitrogen-fixing activity of root nodules in relation to their size in peanut (Arachis hypogaea L.). Plant Production Science 10: 423–429.

Tu, C., Booker, F.L., Burkey, K.O., and Hu, S. 2009. Elevated atmospheric carbon dioxide and O3 differentially alter nitrogen acquisition in peanut. Crop Science 49: 1827–1836.


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