Some nitrogen-fixing bacteria havesymbiotic relationships withplants, especiallylegumes,mosses andaquatic ferns such asAzolla.[4] Looser non-symbiotic relationships between diazotrophs and plants are often referred to as associative, as seen in nitrogen fixation onrice roots. Nitrogen fixation occurs between sometermites andfungi.[5] It occurs naturally in the air by means ofNOx production bylightning.[6][7]
"The protracted investigations of the relation of plants to the acquisition of nitrogen begun byde Saussure,Ville,Lawes,Gilbert and others, and culminated in the discovery of symbiotic fixation by Hellriegel and Wilfarth in 1887."[12]
"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 ofsoil science."[13]
Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by anitrogenase enzyme.[1] The overall reaction for BNF is:
The process is coupled to thehydrolysis of 16 equivalents ofATP and is accompanied by the co-formation of one equivalent ofH 2.[15] The conversion ofN 2 into ammonia occurs at ametal cluster calledFeMoco, an abbreviation for the iron-molybdenum cofactor. The mechanism proceeds via a series ofprotonation and reduction steps wherein the FeMocoactive sitehydrogenates theN 2 substrate.[16] In free-livingdiazotrophs, nitrogenase-generated ammonia is assimilated intoglutamate through theglutamine synthetase/glutamate synthase pathway. The microbialnif 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 inanaerobic conditions, respiring to draw down oxygen levels, or binding the oxygen with aprotein such asleghemoglobin.[21][22]
Atmospheric nitrogen is inaccessible to most organisms,[23] 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,[24] who determined the stoichiometric relationship between C:N:P atoms, The Redfield Ratio, to be 106:16:1.[24]
The protein complex nitrogenase is responsible forcatalyzing the reduction of nitrogen gas (N2) to ammonia (NH3).[25][26] Incyanobacteria, thisenzyme system is housed in a specialized cell called theheterocyst.[27] The production of thenitrogenase 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).[28][29][30] 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.[31] The specialized heterocyst cell is necessary for the performance of nitrogenase as a result of its sensitivity to ambient oxygen.[32]
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, andiron-only, with all three nitrogenase protein variations containing an iron protein component. Molybdenum-dependent nitrogenase is the most commonly present nitrogenase.[33] The different types of nitrogenase can be determined by the specific iron protein component.[34] Nitrogenase is highly conserved.Gene expression throughDNA sequencing can distinguish which protein complex is present in the microorganism and potentially being expressed. Most frequently, thenifH gene is used to identify the presence of molybdenum-dependent nitrogenase, followed by closely related nitrogenase reductases (component II)vnfH andanfH representing vanadium-dependent and iron-only nitrogenase, respectively.[35] In studying the ecology and evolution ofnitrogen-fixing bacteria, thenifH gene is thebiomarker most widely used.[36]nifH has two similar genesanfH and vnfH that also encode for the nitrogenase reductase component of the nitrogenase complex.[37]
Nitrogenase is thought to have evolved sometime between 1.5-2.2 billion years ago (Ga),[38][39] although some isotopic support showing nitrogenase evolution as early as around 3.2 Ga.[40] Nitrogenase appears to have evolved frommaturase-like proteins, although the function of the preceding protein is currently unknown.[41]
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).[42] Marine metal abundances over Earth’s geologic timeline are thought to have driven the relative abundance of which form of nitrogenase was most common.[43] 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 andnitrogen cycle of thebiosphere. In general, cyanobacteria can use various inorganic and organic sources of combined nitrogen, such asnitrate,nitrite,ammonium,urea, or someamino acids. Several cyanobacteria strains are also capable of diazotrophic growth, an ability that may have been present in their last common ancestor in theArchean eon.[48] Nitrogen fixation not only naturally occurs in soils but also aquatic systems, including both freshwater and marine.[49][50] Indeed, the amount of nitrogen fixed in the ocean is at least as much as that on land.[51] The colonial marine cyanobacteriumTrichodesmium is thought to fix nitrogen on such a scale that it accounts for almost half of the nitrogen fixation in marine systems globally.[52] Marine surface lichens and non-photosynthetic bacteria belonging in Proteobacteria and Planctomycetes fixate significant atmospheric nitrogen.[53] Species of nitrogen fixing cyanobacteria in fresh waters include:Aphanizomenon andDolichospermum (previously Anabaena).[54] Such species have specialized cells calledheterocytes, in which nitrogen fixation occurs via the nitrogenase enzyme.[55][56]
Diatoms in the familyRhopalodiaceae also possesscyanobacterialendosymbionts called spheroid bodies or diazoplasts.[60] These endosymbionts have lost photosynthetic properties, but have kept the ability to perform nitrogen fixation, allowing these diatoms to fix atmospheric nitrogen.[61][62] Other diatoms in symbiosis with nitrogen-fixing cyanobacteria are among the generaHemiaulus,Rhizosolenia andChaetoceros.[63]
Plants that contribute to nitrogen fixation include those of thelegumefamily—Fabaceae— withtaxa such askudzu,clover,soybean,alfalfa,lupin,peanut androoibos.[45] They containsymbioticrhizobia bacteria withinnodules in theirroot systems, producing nitrogen compounds that help the plant to grow and compete with other plants.[64] When the plant dies, the fixed nitrogen is released, making it available to other plants; this helps to fertilize thesoil.[21][65] The great majority of legumes have this association, but a fewgenera (e.g.,Styphnolobium) do not. In many traditional farming practices, fields arerotated through various types of crops, which usually include one consisting mainly or entirely ofclover.[citation needed]
Fixation efficiency in soil is dependent on many factors, including thelegume 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).[66]
The ability to fix nitrogen in nodules is present inactinorhizal plants such asalder andbayberry, with the help ofFrankia bacteria. They are found in 25 genera in theordersCucurbitales,Fagales andRosales, which together with theFabales form anitrogen-fixing clade ofeurosids. The ability to fix nitrogen is not universally present in these families. For example, of 122Rosaceae genera, only four fix nitrogen. Fabales were the first lineage to branch off this nitrogen-fixing clade; thus, the ability to fix nitrogen may beplesiomorphic and subsequently lost in most descendants of the original nitrogen-fixing plant; however, it may be that the basicgenetic andphysiological requirements were present in an incipient state in themost recent common ancestors of all these plants, but only evolved to full function in some of them.[67]
In addition,Trema (Parasponia), a tropical genus in the familyCannabaceae, is unusually able to interact with rhizobia and form nitrogen-fixing nodules.[68]
A method for nitrogen fixation was first described byHenry Cavendish in 1784 using electric arcs reacting nitrogen and oxygen in air. This method was implemented in theBirkeland–Eyde process of 1903.[73] 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 byDesfosses in 1828. He observed that mixtures ofalkali metal oxides and carbon react with nitrogen at high temperatures. With the use ofbarium carbonate as starting material, the first commercial process became available in the 1860s, developed by Margueritte and Sourdeval. The resultingbarium cyanide reacts with steam, yielding ammonia. In 1898Frank andCaro developed what is known as theFrank–Caro process to fix nitrogen in the form ofcalcium cyanamide. The process was eclipsed by theHaber process, which was discovered in 1909.[74][75]
Equipment for a study of nitrogen fixation byalpha rays (Fixed Nitrogen Research Laboratory, 1926)
The dominant industrial method for producing ammonia is theHaber process also known as the Haber-Bosch process.[76] Fertilizer production is now the largest source of human-produced fixed nitrogen in the terrestrialecosystem. Ammonia is a required precursor tofertilizers,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[77] 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.[78]
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 givedinitrogen complexes. The first dinitrogencomplex to be reported wasRu(NH 3) 5(N 2)2+.[79] Some soluble complexes do catalyze nitrogen fixation.[80]
Lightning heats the air around it in a high-temperatureplasma, breaking the bonds ofN 2, starting the formation ofnitrous acid (HNO 2).
Nitrogen can be fixed bylightning converting nitrogen gas (N 2) and oxygen gas (O 2) in the atmosphere intoNOx (nitrogen oxides). TheN 2 molecule is highly stable and nonreactive due to thetriple bond between the nitrogen atoms.[81] Lightning produces enough energy and heat to break this bond[81] allowing nitrogen atoms to react with oxygen, formingNO x. These compounds cannot be used by plants, but as this molecule cools, it reacts with oxygen to formNO 2,[82] which in turn reacts with water to produceHNO 2 (nitrous acid) orHNO 3 (nitric acid). When these acids seep into the soil, they makeNO3− (nitrate), which is of use to plants.[83][81]
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