The Nitrogen cycle

Nitrogen cycle Public domain 11/03/11

The nitrogen cycle is the process by which nitrogen is converted between its various chemical forms. This transformation can be carried out via both biological and non-biological processes. Important processes in the nitrogen cycle include fixation, mineralization, nitrification, and denitrification. The majority of Earth's atmosphere (approximately 78%) is nitrogen, making it the largest pool of nitrogen. However, atmospheric nitrogen is unavailable for biological use, leading to a scarcity of usable nitrogen in many types of ecosystems. The nitrogen cycle is of particular interest to ecologists because nitrogen availability can affect the rate of key ecosystem processes, including primary production and decomposition. Human activities such as fossil fuel combustion, use of artificial nitrogen fertilizers, and release of nitrogen in wastewater have dramatically altered the global nitrogen cycle.

Ecological function

Nitrogen is essential for many processes; it is crucial for any life on Earth. It is in all amino acids, is incorporated into proteins, and is present in the bases that make up nucleic acids, such as DNA and RNA. In plants, much of the nitrogen is used in chlorophyll molecules, which are essential for photosynthesis and further growth. Although Earth's atmosphere is an abundant source of nitrogen, most is relatively unusable by plants. Chemical processing, or natural fixation (through processes such as bacterial conversion), are necessary to convert gaseous nitrogen into forms usable by living organisms. This makes nitrogen a crucial part of food production. The abundance or scarcity of this "fixed" form of nitrogen, (also known as reactive nitrogen), dictates how much food can be grown on a piece of land.

The processes of the nitrogen cycle

Nitrogen is present in the environment in a wide variety of chemical forms including organic nitrogen, ammonium (NH4+), nitrate (NO3-), and nitrogen gas (N2). The processes of the nitrogen cycle transform nitrogen from one chemical form to another. Many of the processes are carried out by microbes either to produce energy or to accumulate nitrogen in the form needed for growth. The diagram above shows how these processes fit together to form the nitrogen cycle.

Nitrogen fixation

Atmospheric nitrogen must be processed, or "fixed", to be used by plants. Some fixation occurs in lightning strikes, but most fixation is done by free-living or symbiotic bacteria. These bacteria have the nitrogenase enzyme that combines gaseous nitrogen with hydrogen to produce ammonia, which is then further converted by the bacteria to make their own organic compounds. Most biological nitrogen fixation occurs by the activity of Mo-nitrogenase, found in a wide variety of bacteria and some Archaea. Mo-nitrogenase is a complex two component enzyme that contains multiple metal-containing prosthetic groups.Some nitrogen fixing bacteria, such as Rhizobium, live in the root nodules of legumes (such as peas or beans). Here they form a mutualistic relationship with the plant, producing ammonia in exchange for carbohydrates. Nutrient-poor soils can be planted with legumes to enrich them with nitrogen. A few other plants can form such symbioses. Today, about 30% of the total fixed nitrogen is manufactured in ammonia chemical plants.

Conversion of N2
The conversion of nitrogen (N2) from the atmosphere into a form readily available to plants and hence to animals and humans is an important step in the nitrogen cycle, which distributes the supply of this essential nutrient. There are four ways to convert N2 (atmospheric nitrogen gas) into more chemically reactive forms:

  • Biological fixation: some symbiotic bacteria (most often associated with leguminous plants) and some free-living bacteria are able to fix nitrogen as organic nitrogen. An example of mutualistic nitrogen fixing bacteria are the Rhizobium bacteria, which live in legume root nodules. These species are diazotrophs. An example of the free-living bacteria is Azotobacter.
  • Industrial N-fixation : Under great pressure, at a temperature of 600 C, and with the use of an iron catalyst, atmospheric nitrogen and hydrogen (usually derived from natural gas or petroleum) can be combined to form ammonia (NH3). In the Haber-Bosch process, N2 is converted together with hydrogen gas (H2) into ammonia (NH3), which is used to make fertilizer and explosives.
  • Combustion of fossil fuels : automobile engines and thermal power plants, which release various nitrogen oxides (NOx).
  • Other processes : In addition, the formation of NO from N2 and O2 due to photons and especially lightning, can fix nitrogen.


Some plants get nitrogen from the soil or water, and by absorption of their roots in the form of either nitrate ions or ammonium ions. All nitrogen obtained by animals can be traced back to the eating of plants at some stage of the food chain.

Plants can absorb nitrate or ammonium ions from the soil or water via their root hairs. If nitrate is absorbed, it is first reduced to nitrite ions and then ammonium ions for incorporation into amino acids, nucleic acids, and chlorophyll. In plants that have a mutualistic relationship with rhizobia, some nitrogen is assimilated in the form of ammonium ions directly from the nodules. Animals, fungi, and other heterotrophic organisms absorb nitrogen as amino acids, nucleotides and other small organic molecules.


When a plant dies, an animal dies, or an animal expels waste, the initial form of nitrogen is organic. Bacteria, or in some cases, fungi, convert the organic nitrogen within the remains back into ammonium (NH4+), a process called ammonification or mineralization. Enzymes Involved:

  • GS: Gln Synthetase (Cytosolic & PLastid)
  • GOGAT: Glu 2-oxoglutarate aminotransferase (Ferredoxin & NADH dependent)
  • GDH: Glu Dehydrogenase:
    • o Minor Role in ammonium assimilation.
    • o Important in amino acid catabolism.


Nitrification is the biological oxidation of ammonia with oxygen into nitrite followed by the oxidation of these nitrites into nitrates. Degradation of ammonia to nitrite is usually the rate limiting step of nitrification. Nitrification is an important step in the nitrogen cycle in water and soil. This process was discovered by the Russian microbiologist, Sergei Winogradsky. The primary stage of nitrification, the oxidation of ammonium (NH4+) is performed by bacteria such as the Nitrosomonas species, which converts ammonia to nitrites (NO2-). Other bacterial species, such as the Nitrobacter, are responsible for the oxidation of the nitrites into nitrates (NO3-). It is important for the nitrites to be converted to nitrates because accumulated nitrites are toxic to plant life.

Microbiology and ecology
The oxidation of ammonia into nitrite is performed by two groups of organisms, ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea(AOA). Currently, only one ammonia-oxidizing archaeon, Nitrosopumilus maritimus, has been isolated and described. In soils the most studied ammonia oxidizing bacteria belong to the genera Nitrosomonas and Nitrosococcus. Although in soils ammonia oxidation occurs by both bacteria and archaea, archaeal ammonia oxidizers dominate in both soils and marine environments, suggesting that Crenarchaeota may be greater contributors to ammonia oxidation in these environments. The second step (oxidation of nitrite into nitrate) is done (mainly) by bacteria of the genus Nitrobacter. Both steps are producing energy to be coupled to ATP synthesis. Nitrifying organisms are chemoautotrophs, and use carbon dioxide as their carbon source for growth. Some AOB possess the enzyme, urease, which catalyzes the conversion of the urea molecule to two ammonia molecules and one carbon dioxide molecule. Nitrosomonas europaea, as well as populations of soil-dwelling AOB, have been shown to assimilate the carbon dioxide released by the reaction to make biomass via the Calvin Cycle, and harvest energy by oxidizing ammonia (the other product of urease) to nitrite. This feature may explain enhanced growth of AOB in the presence of urea in acidic environments.

Nitrification also plays an important role in the removal of nitrogen from municipal wastewater. The conventional removal is nitrification, followed by denitrification. The cost of this process resides mainly in aeration (bringing oxygen in the reactor) and the addition of an external carbon source (e.g., methanol) for the denitrification.

Due to their very high solubility, nitrates can enter groundwater. In distribution systems where chloramines are used as the secondary disinfectant, the presence of free ammonia can act as a substrate for ammonia-oxidizing microorganisms. The associated reactions can lead to the depletion of the disinfectant residual in the system. Elevated nitrate in groundwater is a concern for drinking water use because nitrate can interfere with blood-oxygen levels in infants and cause methemoglobinemia or blue-baby syndrome. Where groundwater recharges stream flow, nitrate-enriched groundwater can contribute to eutrophication, a process leading to high algal, especially blue-green algal populations and the death of aquatic life due to excessive demand for oxygen. While not directly toxic to fish life like ammonia, nitrate can have indirect effects on fish if it contributes to this eutrophication. Nitrogen has contributed to severe eutrophication problems in some water bodies. As of 2006, the application of nitrogen fertilizer is being increasingly controlled in Britain and the United States. This is occurring along the same lines as control of phosphorus fertilizer, restriction of which is normally considered essential to the recovery of eutrophied waterbodies.

In most environments, both organisms are found together, yielding nitrate as the final product. However, it is possible to design systems in which nitrite is formed (the Sharon process).

Together with ammonification, nitrification forms a mineralization process that refers to the complete decomposition of organic material, with the release of available nitrogen compounds. This replenishes the nitrogen cycle.

Nitrification is a process of nitrogen compound oxidation (effectively, loss of electrons from the nitrogen atom to the oxygen atoms):

  • NH3 + CO2 + 1.5 O2 + Nitrosomonas ? NO2- + H2O + H+
  • NO2- + CO2 + 0.5 O2 + Nitrobacter ? NO3-
  • NH3 + O2 ? NO2- + 3H+ + 2e-
  • NO2- + H2O ? NO3- + 2H+ + 2e-


Denitrification is a microbially facilitated process of nitrate reduction that may ultimately produce molecular nitrogen (N2) through a series of intermediate gaseous nitrogen oxide products. This process is performed by bacterial species such as Pseudomonas and Clostridium in anaerobic conditions. They use the nitrate as an electron acceptor in the place of oxygen during respiration. These facultatively anaerobic bacteria can also live in aerobic conditions. This respiratory process reduces oxidized forms of nitrogen in response to the oxidation of an electron donor such as organic matter. The preferred nitrogen electron acceptors in order of most to least thermodynamically favorable include nitrate (NO3-), nitrite (NO2-), nitric oxide (NO), and nitrous oxide (N2O). In terms of the general nitrogen cycle, denitrification completes the cycle by returning N2 to the atmosphere. The process is performed primarily by heterotrophic bacteria (such as Paracoccus denitrificans and various pseudomonads), although autotrophic denitrifiers have also been identified (e.g., Thiobacillus denitrificans). Denitrifiers are represented in all main phylogenetic groups. Generally several species of bacteria are involved in the complete reduction of nitrate to molecular nitrogen, and more than one enzymatic pathway have been identified in the reduction process.

Direct reduction from nitrate to ammonium, a process known as dissimilatory nitrate reduction to ammonium or DNRA, is also possible for organisms that have the nrf-gene. This is less common than denitrification in most ecosystems as a means of nitrate reduction. Other genes known in microorganisms which denitrify include nir (nitrite reductase) and nos (nitrous oxide reductase) among others; organisms identified as having these genes include Alcaligenes faecalis, Alcaligenes xylosoxidans, many in the Pseudomonas genus, Bradyrhizobium japonicum, and Blastobacter denitrificans.

Nutrient limitation
All organisms require certain nutrients in their surroundings (available to them) for survival. Depending upon the ecosystem, nitrogen is most likely the limiting nutrient, although phosphorus is the other primary limiting nutrient and these two elements interact chemically. Some organisms appear to be able to denitrify and remove phosphorus. The triple bond of N2 makes this a very stable compound; most organisms (i.e. plants) depend upon others to break this down to make it available for biochemical reactions. See Nitrification. Symbiotic relationships between Rhizobium species and legumes are well-documented.

Conditions required
Denitrification takes place under special conditions in both terrestrial and marine ecosystems. In general, it occurs where oxygen, a more energetically favourable electron acceptor, is depleted, and bacteria respire nitrate as a substitute terminal electron acceptor. Due to the high concentration of oxygen in our atmosphere, denitrification only takes place in environments where oxygen consumption exceeds the rate of oxygen supply, such as in some soils and groundwater, wetlands, poorly ventilated corners of the ocean, and in seafloor sediments.

Denitrification generally proceeds through some combination of the following intermediate forms:

  • NO3- ? NO2- ? NO + N2O ? N2 (g)

The complete denitrification process can be expressed as a redox reaction:

  • 2 NO3- + 10 e- + 12 H+ ? N2 + 6 H2O

This reaction shows a fractionation in isotope composition. Lighter isotopes of nitrogen are preferred in the reaction, leaving the heavier nitrogen isotopes in the residual matter. The process can cause delta-values of up to -40, where delta is a representation of the difference in isotopic composition. This can be used to identify denitrification processes in nature.

Denitrification by rhizobia
Rhizobia are soil bacteria with the unique ability to establish a N2-fixing symbiosis on legume roots. When faced with a shortage of oxygen some rhizobia species are able to switch from O2-respiration to using nitrates to support respiration. This denitrification pathway comprises the sequential reduction of nitrate or nitrite to dinitrogen, via the gaseous intermediates nitric oxide and nitrous oxide. The enzymes involved in denitrification are nitrate-, nitrite-, nitric oxide- and nitrous oxide reductase, encoded by nar/nap, nir, nor and nos genes, respectively. In recent years it has emerged that many rhizobia species have genes for enzymes of some or all of the four reductase reactions for denitrification. In fact, denitrification can be readily observed in many rhizobia species, in their free-living form, in legume root nodules, or in isolated bacteroids. This chapter will focus on update progress on denitrification by rhizobia under free-living and symbiotic conditions.

Deliberate use of process
Denitrification is commonly used to remove nitrogen from sewage and municipal wastewater. It is also an instrumental process in wetlands and riparian zones for the removal of excess nitrate from groundwater resulting from excessive agricultural or residential fertilizer usage.

Reduction under anoxic conditions can also occur through process called anaerobic ammonia oxidation (anammox):

  • NH4+ + NO2- ? N2 + 2 H2O

In some wastewater treatment plants, small amounts of methanol, ethanol, acetate or proprietary products like MicroCg or MicroCglycerin are added to the wastewater to provide a carbon source for the denitrification bacteria. Denitrification processes are also used in the treatment of industrial wastes.

Influence on global climate change
Increasing carbon dioxide levels within the atmosphere will influence global nutrient cycling, yet it is difficult to predict what those interactions might be.Chemical interactions between soils and the atmosphere will be influenced by changes in atmospheric composition. There are indications that increased fertilization of soils with nitrogen causes a decrease in carbon sequestration.

Jake Beaulieu, a postdoctoral researcher the Environmental Protection Agency in Cincinnati, Ohio and Jennifer Tank, Galla Professor of Biological Sciences at the University of Notre Dame, are lead authors of new paper demonstrating that streams and rivers receiving nitrogen inputs from urban and agricultural land uses are a significant source of nitrous oxide to the atmosphere.

Anaerobic ammonium oxidation

In this biological process, nitrite and ammonium are converted directly into dinitrogen gas. This process makes up a major proportion of dinitrogen conversion in the oceans.

Anammox, an abbreviation for ANaerobic AMMonium OXidation, is a globally important microbial process of the nitrogen cycle. The bacteria mediating this process were identified only 20 years ago and at the time were a great surprise for the scientific community. It takes place in many natural environments and anammox is also the trademarked name for an ammonium removal technology that has been developed by the Delft University of Technology.

Process background
In this biological process, nitrite and ammonium are converted directly into dinitrogen gas. This process contributes up to 50% of the dinitrogen gas produced in the oceans. It is thus a major sink for fixed nitrogen and so limits oceanic primary productivity. The overall catabolic reaction is:

  • NH4+ + NO2- ? N2 + 2H2O

The bacteria that perform the anammox process belong to the bacterial phylum Planctomycetes, of which Planctomyces and Pirellula are the best known genera. Currently four genera of anammox bacteria have been (provisionally) defined: Brocadia, Kuenenia, Anammoxoglobus, Jettenia (all fresh water species), and Scalindua (marine species). The anammox bacteria are characterized by several striking properties: they all possess one anammoxosome, a membrane bound compartment inside the cytoplasm which is the locus of anammox catabolism. Further, the membranes of these bacteria mainly consist of ladderane lipids so far unique in Biology. Of special interest is the turnover of hydrazine (normally used as a high-energy rocket fuel, and poisonous to most living organisms) as an intermediate. A final striking feature of the organism is the extremely slow growth rate. The doubling time is nearly two weeks. The anammox process was originally found to occur only from 20°C to 43°C but more recently, anammox has been observed at temperatures from 36°C to 52°C in hot springs and 60oC to 85oC at hydrothermal vents located along the Mid-Atlantic Ridge.

For a long time the general consensus was that ammonium could only be oxidised under aerobic conditions. The Austrian theoretical chemist Engelbert Broda was the first to recognise the possibility of anaerobic ammonium oxidation in 1977. The simultaneous removal of ammonium and production of nitrogen gas was observed in an industrial wastewater treatment in The Netherlands in 1986.

The application of the anammox process lies in the removal of ammonium in wastewater treatment and consists of two separate processes. The first step is partial nitrification (nitritation) of half of the ammonium to nitrite by ammonia oxidizing bacteria:

  • 4NH4+ + 3O2 ? 2NH4+ + 2NO2- + 4H+ + 2H2O

The resulting ammonium and nitrite are converted in the anammox process to dinitrogen gas and circa 15% nitrate (not shown) by anammox bacteria

  • NH4+ + NO2- ? N2 + 2 H2O

Both processes can take place in 1 reactor where two guilds of bacteria form compact granules.

For the enrichment of the anammox organisms a granular biomass or biofilm system seems to be especially suited in which the necessary sludge age of more than 20 days can be ensured. Possible reactors are sequencing batch reactors (SBR), moving bed reactors or gas-lift-loop reactors. The cost reduction compared to conventional nitrogen removal is considerable; the technique is still young but proven in several fullscale installations. The first full scale reactor intended for the application of anammox bacteria was built in the Netherlands in 2002. Other wastewater treatment plants, such as the one in in Germany (Hattingen), where anammox activity is coincidentally observed were not purpose built. As of 2006 there are three full scale processes in The Netherlands. One on a municipal wastewater treatment plant (in Rotterdam), and two on industrial effluent. One is a tannery, the other a potato processing plant.

Human influences on the nitrogen cycle

As a result of extensive cultivation of legumes (particularly soy, alfalfa, and clover), growing use of the Haber-Bosch process in the creation of chemical fertilizers, and pollution emitted by vehicles and industrial plants, human beings have more than doubled the annual transfer of nitrogen into biologically-available forms. In addition, humans have significantly contributed to the transfer of nitrogen trace gases from Earth to the atmosphere, and from the land to aquatic systems. Human alterations to the global nitrogen cycle are most intense in developed countries and in Asia, where vehicle emissions and industrial agriculture are highest.

N2O (nitrous oxide) has risen in the atmosphere as a result of agricultural fertilization, biomass burning, cattle and feedlots, and other industrial sources. N2O has deleterious effects in the stratosphere, where it breaks down and acts as a catalyst in the destruction of atmospheric ozone.

N2O in the atmosphere is a greenhouse gas, currently the third largest contributor to global warming, after carbon dioxide and methane. While not as abundant in the atmosphere as carbon dioxide, for an equivalent mass, nitrous oxide is nearly 300 times more potent in its ability to warm the planet.

NH3 (ammonia) in the atmosphere has tripled as the result of human activities. It is a reactant in the atmosphere, where it acts as an aerosol, decreasing air quality and clinging on to water droplets, eventually resulting in nitric acid (HNO3) acid rain. Atmospheric NH3 and HNO3 damage respiratory systems.

All forms of high-temperature combustion have contributed to a 6 or 7 fold increase in NOx flux to the atmosphere. It is a function of combustion temperature - the higher the temperature, the more NOx is produced. Fossil fuel combustion is a primary contributor, but so are biofuels and even burning hydrogen. The higher combustion temperature of hydrogen produces more NOx than natural gas combustion. The very-high temperature of lightning produces small amounts of NOx, NH3, and HNO3.

NH3 and NOx actively alter atmospheric chemistry. They are precursors of tropospheric (lower atmosphere) ozone production, which contributes to smog, acid rain, damages plants and increases nitrogen inputs to ecosystems. Ecosystem processes can increase with nitrogen fertilization, but anthropogenic input can also result in nitrogen saturation, which weakens productivity and can damage the health of plants, animals, fish, and humans.

Decreases in biodiversity can also result if higher nitrogen availability increases nitrogen-demanding grasses, causing a degradation of nitrogen-poor, species diverse heathlands.

Wastewater treatment
Onsite sewage facilities such as septic tanks and holding tanks release large amounts of nitrogen into the environment by discharging through a drainfield into the ground. Microbial activity consumes the nitrogen and other contaminants in the wastewater.

However, in certain areas, the soil is unsuitable to handle some or all of the wastewater, and, as a result, the wastewater with the contaminants enters the aquifers. These contaminants accumulate and eventually end up in drinking water. One of the contaminants concerned about the most is nitrogen in the form of nitrates. A nitrate concentration of 10 ppm (parts per million) or 10 milligrams per liter is the current EPA limit for drinking water and typical household wastewater can produce a range of 20 - 85 ppm.

The health risk associated with drinking water (with >10 ppm nitrate) is the development of methemoglobinemia and has been found to cause blue baby syndrome. Several states have now started programs to introduce advanced wastewater treatment systems to the typical onsite sewage facilities. The result of these systems is an overall reduction of nitrogen, as well as other contaminants in the wastewater.

Environmental impacts
Additional risks posed by increased availability of inorganic nitrogen in aquatic ecosystems include water acidification; eutrophication of fresh and saltwater systems; and toxicity issues for animals, including humans. Eutrophication often leads to lower dissolved oxygen levels in the water column, including hypoxic and anoxic conditions, which can cause cause death of aquatic fauna. Relatively sessile benthos, or bottom-dwelling creatures, are particularly vulnerable because of their lack of mobility, though large fish kills are not uncommon. Oceanic dead zones near the mouth of the Mississippi in the Gulf of Mexico are a well-known examples of algal bloom-induced hypoxia.

The New York Adirondack Lakes, Catskills, Hudson Highlands, Rensselaer Plateau and parts of Long Island are examples of the impact of nitric acid raid deposition, killing fish and many other aquatic species.

Ammonia (NH3) is highly toxic to fish and the water discharge level of ammonia from wastewater treatment facilities must often be closely monitored. To prevent fish deaths, nitrification prior to discharge is often desirable. Land application can be an attractive alternative to the mechanical aeration needed for nitrification.