Conversion of molecular nitrogen into biologically accessible nitrogen compounds
Nitrogen fixation is a chemical process by which molecular
nitrogen (N 2), which has a strong triple
covalent bond, is converted into
ammonia (NH 3) or related nitrogenous compounds, typically in soil or aquatic systems[1] but also
in industry. The nitrogen in air 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 microbe-mediated process that converts
dinitrogen (N2) gas to ammonia (NH3) using the
nitrogenase protein complex (Nif).[2][3]
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 include some explosives, pharmaceuticals, and dyes.
Nitrogen fixation is carried out naturally in
soil by
microorganisms termed
diazotrophs that include
bacteria, such as Azotobacter and Rhizobia, and
archaea. Some nitrogen-fixing bacteria have
symbiotic relationships with plant groups, especially
legumes.[4] 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.[5] It occurs naturally in the air by means of
NOx production by
lightning.[6][7]
All biological reactions involving the process of nitrogen fixation are catalyzed by enzymes called
nitrogenases.[8] These enzymes contain
iron, often with a second metal, usually
molybdenum but sometimes
vanadium.
"The protracted investigations of the relation of plants to the acquisition of nitrogen begun by
de Saussure,
Ville,
Lawes,
Gilbert and others, and culminated in the discovery of symbiotic fixation by Hellriegel and Wilfarth in 1887."[13]
"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 1886-1888 would open a new era of
soil science."[14]
Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by a
nitrogenase enzyme.[1] The overall reaction for BNF is:
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.[15] 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 sitehydrogenates the N 2 substrate.[16] 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.[17]
For example, decomposing wood, which generally has a low nitrogen content, has been shown to host a diazotrophic community.[18][19] The bacteria enrich the wood substrate with nitrogen through fixation, thus enabling deadwood decomposition by fungi.[20]
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.[1]
Atmospheric nitrogen is inaccessible to most organisms,[21] because its triple covalent bond is very strong. Most take up fixed nitrogen from various sources. 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,[22] who determined the stoichiometric relationship between C:N:P atoms, The Redfield Ratio, to be 106:16:1.[22]
The protein complex nitrogenase is responsible for
catalyzing the reduction of nitrogen gas (N2) to ammonia (NH3).[23] In
cyanobacteria, this
enzyme system is housed in a specialized cell called the
heterocyst.[24] 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).[25][26][27] 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.[28] The specialized heterocyst cell is necessary for the performance of nitrogenase as a result of its sensitivity to ambient oxygen.[29]
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 protein variations containing an iron protein component. Molybdenum-dependent nitrogenase is the most commonly present nitrogenase.[23] The different types of nitrogenase can be determined by the specific iron protein component.[30] Nitrogenase is highly conserved.
Gene expression through
DNA sequencing can distinguish which protein complex is present in the microorganism and potentially being expressed. 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.[31] In studying the ecology and evolution of
nitrogen-fixing bacteria, the nifH gene is the
biomarker most widely used.[32]nifH has two similar genes anfH and vnfH that also encode for the nitrogenase reductase component of the nitrogenase complex.[33]
Evolution of Nitrogenase
The origin of nitrogenase has been of interest to paleobiologists and is an area of active research.[34] Nitrogenase is thought to have evolved sometime between 1.5-2.2 billion years ago (Ga)[35] although some isotopic support showing nitrogenase evolution as early as around 3.2 Ga.[36] Nitrogenase appears to have evolved from maturase-like proteins, although the function of the preceding protein is currently unknown.[37]
Nitrogenase has three different forms (Nif, Anf, and Vnf) that correspond with the metal found in the active site of the protein (Molybdenum, Iron, and Vanadium respectively).[38] Marine metal abundances over Earth’s geologic timeline are thought to have driven the relative abundance of which form of nitrogenase was most common.[39] Currently, there is no conclusive agreement on which form of nitrogenase arose first.
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.[44] Nitrogen fixation not only naturally occurs in soils but also aquatic systems, including both freshwater and marine.[45][46] Indeed, the amount of nitrogen fixed in the ocean is at least as much as that on land.[47] 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.[48] Marine surface lichens and non-photosynthetic bacteria belonging in Proteobacteria and Planctomycetes fixate significant atmospheric nitrogen.[49] Species of nitrogen fixing cyanobacteria in fresh waters include: Aphanizomenon and
Dolichospermum (previously Anabaena).[50] Such species have specialized cells called
heterocytes, in which nitrogen fixation occurs via the nitrogenase enzyme.[51][52]
Algae
One type of
organelle can turn nitrogen gas into a biologically available form. This
nitroplast was discovered in
algae.[53]
Plants that contribute to nitrogen fixation include those of the
legumefamily—
Fabaceae— with
taxa such as
kudzu,
clover,
soybean,
alfalfa,
lupin,
peanut and
rooibos.[41] They contain
symbioticrhizobia bacteria within
nodules in their
root systems, producing nitrogen compounds that help the plant to grow and compete with other plants.[54] When the plant dies, the fixed nitrogen is released, making it available to other plants; this helps to fertilize the
soil.[1][55] 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.[citation needed]
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 (56 to 224 kg/ha).[56]
Non-leguminous
The ability to fix nitrogen in nodules is present in
actinorhizal plants such as
alder and
bayberry, with the help of Frankia bacteria. They are found in 25 genera in the
ordersCucurbitales,
Fagales and
Rosales, which together with the
Fabales form a nitrogen-fixing 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.[57]
In addition, Trema (Parasponia), a tropical genus in the family
Cannabaceae, is unusually able to interact with rhizobia and form nitrogen-fixing nodules.[58]
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 of 1903.[63] The fixation of nitrogen by lightning is a very similar natural occurring process.
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 with nitrogen at high temperatures. 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. In 1898
Frank and
Caro developed what is known as the
Frank–Caro process to fix nitrogen in the form of
calcium cyanamide. The process was eclipsed by the
Haber process, which was discovered in 1909.[64][65]
The dominant industrial method for producing ammonia is the
Haber process also known as the Haber-Bosch process.[66] 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 product has resulted in an intensification of nitrogen fertilizer globally[67] and is credited with supporting the expansion of the human population from around 2 billion in the early 20th century to roughly 8 billion people now.[68]
Much research has been conducted on the discovery of catalysts for nitrogen fixation, often with the goal of lowering 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(N 2)2+.[69] Some soluble complexes do catalyze nitrogen fixation.[70]
Lightning
Nitrogen can be fixed by
lightning converting nitrogen gas (N 2) and oxygen gas (O 2) in the atmosphere into NOx (
nitrogen oxides). The N 2 molecule is highly stable and nonreactive due to the
triple bond between the nitrogen atoms.[71] Lightning produces enough energy and heat to break this bond[71] 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,[72] which in turn reacts with water to produce HNO 2 (
nitrous acid) or HNO 3 (
nitric acid). When these acids seep into the soil, they make
NO 3 (nitrate), which is of use to plants.[73][71]
^Wagner SC (2011).
"Biological Nitrogen Fixation". Nature Education Knowledge. 3 (10): 15.
Archived from the original on 13 September 2018. Retrieved 29 January 2019.
^Lee CC, Ribbe MW, Hu Y (2014). Kroneck PM, Sosa Torres ME (eds.). "Chapter 7. Cleaving the N,N Triple Bond: The Transformation of Dinitrogen to Ammonia by Nitrogenases". Metal Ions in Life Sciences. 14. Springer: 147–76.
doi:
10.1007/978-94-017-9269-1_7.
PMID25416394.
^Delwiche CC (1983). "Cycling of Elements in the Biosphere". In Läuchli A, Bieleski RL (eds.). Inorganic Plant Nutrition. Encyclopedia of Plant Physiology. Berlin, Heidelberg: Springer. pp. 212–238.
doi:
10.1007/978-3-642-68885-0_8.
ISBN978-3-642-68885-0.
^Wolk CP, Ernst A, Elhai J (1994). "Heterocyst Metabolism and Development". In Bryant DA (ed.). The Molecular Biology of Cyanobacteria. Advances in Photosynthesis. Dordrecht: Springer Netherlands. pp. 769–823.
doi:
10.1007/978-94-011-0227-8_27.
ISBN978-94-011-0227-8.
^Schneider K, Müller A (2004). "Iron-Only Nitrogenase: Exceptional Catalytic, Structural and Spectroscopic Features". In Smith BE, Richards RL, Newton WE (eds.). Catalysts for Nitrogen Fixation. Nitrogen Fixation: Origins, Applications, and Research Progress. Dordrecht: Springer Netherlands. pp. 281–307.
doi:
10.1007/978-1-4020-3611-8_11.
ISBN978-1-4020-3611-8.
^Schüddekopf K, Hennecke S, Liese U, Kutsche M, Klipp W (May 1993). "Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus". Molecular Microbiology. 8 (4): 673–684.
doi:
10.1111/j.1365-2958.1993.tb01611.x.
PMID8332060.
S2CID42057860.
^Dawson JO (2008). "Ecology of Actinorhizal Plants". Nitrogen-fixing Actinorhizal Symbioses. Nitrogen Fixation: Origins, Applications, and Research Progress. Vol. 6. Springer. pp. 199–234.
doi:
10.1007/978-1-4020-3547-0_8.
ISBN978-1-4020-3540-1.
^Eyde S (1909). "The Manufacture of Nitrates from the Atmosphere by the Electric Arc—Birkeland-Eyde Process". Journal of the Royal Society of Arts. 57 (2949): 568–576.
JSTOR41338647.