Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by a
nitrogenase enzyme. 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 cannot be metabolized by most organisms, 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, who determined the stoichiometric relationship between C:N:P atoms, The Redfield Ratio, to be 106:16:1. In
cyanobacteria, this
enzyme system is housed in a specialized 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). Three iron-dependent proteins are known:
molybdenum-dependent,
vanadium-dependent, and
iron-only, with all three nitrogenase protein variations containing an iron protein component. Molybdenum-dependent nitrogenase is most common. 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. 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.
Evolution of nitrogenase Nitrogenase is thought to have evolved sometime between 1.5-2.2 billion years ago (Ga), although there is some isotopic support for nitrogenase evolution as early as around 3.2 Ga. Nitrogenase appears to have evolved from
maturase-like proteins, although the function of the preceding protein is currently unknown. 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). Marine metal abundances over Earth's geologic timeline are thought to have driven the relative abundance of which form of nitrogenase was most common. Currently, there is no conclusive agreement on which form of nitrogenase arose first.
Microorganisms Diazotrophs are widespread within domain
Bacteria including
cyanobacteria (e.g. the highly significant
Trichodesmium and
Cyanothece),
green sulfur bacteria,
purple sulfur bacteria,
Azotobacteraceae,
rhizobia and
Frankia. Several obligately anaerobic bacteria fix nitrogen including many (but not all)
Clostridium spp. Some
archaea such as
Methanosarcina acetivorans also fix nitrogen, and several other
methanogenic
taxa, 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. Indeed, the amount of nitrogen fixed in the ocean is at least as much as that on land. 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. Marine surface lichens and non-photosynthetic bacteria belonging in Proteobacteria and Planctomycetes fixate significant atmospheric nitrogen. 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.
Algae One type of
organelle, originating from
cyanobacterial
endosymbionts called
UCYN-A2, can turn nitrogen gas into a biologically available form. This
nitroplast was discovered in
algae, particularly in the marine algae
Braarudosphaera bigelowii.
Diatoms in the family
Rhopalodiaceae also possess
cyanobacterial
endosymbionts called spheroid bodies or diazoplasts. These endosymbionts have lost photosynthetic properties, but have kept the ability to perform nitrogen fixation, allowing these diatoms to fix atmospheric nitrogen. Other diatoms in symbiosis with nitrogen-fixing cyanobacteria are among the genera
Hemiaulus,
Rhizosolenia and
Chaetoceros.
Root nodule symbioses Legume family 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. 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 .
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
orders
Cucurbitales,
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. In addition,
Trema (
Parasponia), a tropical genus in the family
Cannabaceae, is unusually able to interact with rhizobia and form nitrogen-fixing nodules.
Other plant symbionts Some other plants live in association with a
cyanobiont (cyanobacteria such as
Nostoc) which fix nitrogen for them: • Some lichens such as
Lobaria and
Peltigera •
Mosquito fern (
Azolla species) •
Cycads •
Gunnera •
Blasia (
liverwort) •
Hornworts Some symbiotic relationships involving agriculturally-important plants are: •
Sugarcane and unclear
endophytes •
Foxtail millet and
Azospirillum brasilense •
Kallar grass and
Azoarcus sp. strain BH72 •
Rice and
Herbaspirillum seropedicae •
Wheat and
Klebsiella pneumoniae •
Maize landrace '
Sierra Mixe' / 'olotón' and various
Bacteroidota and
Pseudomonadota == Industrial processes ==