Nitrification is the biological oxidation of ammonia or ammonium to nitrite followed by the oxidation of the nitrite to nitrate. The transformation of ammonia to nitrite is usually the rate limiting step of nitrification. Nitrification is an important step in the nitrogen cycle in soil. Nitrification is an aerobic process performed by small groups of autotrophic bacteria and archaea. This process was discovered by the Russian microbiologist Sergei Winogradsky.
The oxidation of ammonia into nitrite is performed by two groups of organisms, ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA). AOB can be found among the β-proteobacteria and gammaproteobacteria. Currently, two AOA, Nitrosopumilus maritimus and Nitrososphaera viennensis, have been isolated and described. In soils the most studied AOB belong to the genera Nitrosomonas and Nitrosococcus. Although in soils ammonia oxidation occurs by both AOB and AOA, AOA dominate in both soils and marine environments, suggesting that Thaumarchaeota 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 and Nitrospira. 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.
In most environments, organisms are present that will complete both steps of the process, yielding nitrate as the final product. However, it is possible to design systems in which nitrite is formed (the Sharon process).
Nitrification is important in agricultural systems, where fertilizer is often applied as ammonia. Conversion of this ammonia to nitrate increases nitrogen leaching because nitrate is more water-soluble than ammonia.
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.
Nitrification can also occur in drinking water. 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. The addition of chlorite ion to chloramine-treated water has been shown to control nitrification.
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.
Another as-of-yet unknown enzyme that converts nitric oxide to nitrite.
The third step (nitrite to nitrate) is completed in a different organism.
In the marine environment, nitrogen is often the limiting nutrient, so the nitrogen cycle in the ocean is of particular interest. The nitrification step of the cycle is of particular interest in the ocean because it creates nitrate, the primary form of nitrogen responsible for "new" production. Furthermore, as the ocean becomes enriched in anthropogenic CO2, the resulting decrease in pH could lead to decreasing rates of nitrification. Nitrification could potentially become a "bottleneck" in the nitrogen cycle.
Nitrification, as stated above, is formally a two-step process; in the first step ammonia is oxidized to nitrite, and in the second step nitrite is oxidized to nitrate. Different microbes are responsible for each step in the marine environment. Several groups of ammonia-oxidizing bacteria (AOB) are known in the marine environment, including Nitrosomonas, Nitrospira, and Nitrosococcus. All contain the functional gene ammonia monooxygenase (AMO) which, as its name implies, is responsible for the oxidation of ammonia. More recent metagenomic studies have revealed that some Thaumarchaeota (formerly Crenarchaeota) possess AMO. Thaumarchaeotes are abundant in the ocean and some species have a 200 times greater affinity for ammonia than AOB, leading researchers to challenge the previous belief that AOB are primarily responsible for nitrification in the ocean. Furthermore, though nitrification is classically thought to be vertically separated from primary production because the oxidation of nitrogen by bacteria is inhibited by light, nitrification by AOA does not appear to be light inhibited, meaning that nitrification is occurring throughout the water column, challenging the classical definitions of "new" and "recycled" production.
In the second step, nitrite is oxidized to nitrate. In the oceans, this step is not as well understood as the first, but the bacteria Nitrospina and Nitrobacter are known to carry out this step in the sea.
Nitrification inhibitors are chemical compounds that slow the nitrification of ammonia, ammonium-containing, or urea-containing fertilizers, which are applied to soil as fertilizers. These inhibitors can help reduce losses of nitrogen in soil that would otherwise be used by crops. Nitrification inhibitors are used widely, being added to approximately 50% of the fall-applied anhydrous ammonia in states in the U.S., like Illinois. They are usually effective in increasing recovery of nitrogen fertilizer in row crops, but the level of effectiveness depends on external conditions and their benefits are most likely to be seen at less than optimal nitrogen rates.
The environmental concerns of nitrification also contribute to interest in the use of nitrification inhibitors: the primary product, nitrate, leaches into groundwater, producing acute toxicity in multiple species of wildlife and contributing to the eutrophication of standing water. Some inhibitors of nitrification also inhibit the production of methane, a greenhouse gas.
The inhibition of the nitrification process is primarily facilitated by the selection and inhibition/destruction of the bacteria that oxidize ammonia compounds. A multitude of compounds that inhibit nitrification, which can be divided into the following areas: the active site of ammonia monooxygenase (AMO), mechanistic inhibitors, and the process of N-heterocyclic compounds. The process for the latter of the three is not yet widely understood, but is prominent. The presence of AMO has been confirmed on many substrates that are nitrogen inhibitors such as dicyandiamide, ammonium thiosulfate, and nitrapyrin.
The conversion of ammonia to hydroxylamine is the first step in nitrification, where AH2 represents a range of potential electron donors.
This reaction is catalyzed by AMO. Inhibitors of this reaction bind to the active site on AMO and prevent or delay the process. The process of oxidation of ammonia by AMO is regarded with importance due to the fact that other processes require the co-oxidation of NH3 for a supply of reducing equivalents. This is usually supplied by the compound hydroxylamine oxidoreductase (HAO) which catalyzes the reaction:
The mechanism of inhibition is complicated by this requirement. Kinetic analysis of the inhibition of NH3 oxidation has shown that the substrates of AMO have shown kinetics ranging from competitive to noncompetitive. The binding and oxidation can occur on two different locations on AMO: in competitive substrates, binding and oxidation occurs at the NH3 site, while in noncompetitive substrates it occurs at another site.
Mechanism based inhibitors can be defined as compounds that interrupt the normal reaction catalyzed by an enzyme. This method occurs by the inactivation of the enzyme via covalent modification of the product, which ultimately inhibits nitrification. Through the process, AMO is deactivated and one or more proteins is covalently bonded to the final product. This is found to be most prominent in a broad range of sulfur or acetylenic compounds.
Sulfur-containing compounds, including ammonium thiosulfate (a popular inhibitor) are found to operate by producing volatile compounds with strong inhibitory effects such as carbon disulfide and thiourea.
In particular, thiophosphoryl triamide has been a notable addition where it has the dual purpose of inhibiting both the production of urease and nitrification. In a study of inhibitory effects of oxidation by the bacteria Nitrosomonas europaea, the use of thioethers resulted in the oxidation of these compounds to sulfoxides, where the S atom is the primary site of oxidation by AMO. This is most strongly correlated to the field of competitive inhibition.
N-heterocyclic compounds are also highly effective nitrification inhibitors and are often classified by their ring structure. The mode of action for these compounds is not well understood: while nitrapyrin, a widely used inhibitor and substrate of AMO, is a weak mechanism-based inhibitor of said enzyme, the effects of said mechanism are unable to correlate directly with the compound’s ability to inhibit nitrification. It is suggested that nitrapyrin acts against the monooxygenase enzyme within the bacteria, preventing growth and CH4/NH4 oxidation. Compounds containing two or three adjacent ring N atoms (pyridazine, pyrazole, indazole) tend to have a significantly higher inhibition effect than compounds containing non-adjacent N atoms or singular ring N atoms (pyridine, pyrrole). This suggests that the presence of ring N atoms is directly correlated with the inhibition effect of this class of compounds.
Some enzymatic nitrification inhibitors, such as urease, can also inhibit the production of methane in methanotrophic bacteria. AMO shows similar kinetic turnover rates to methane monooxygenase (MMO) found in methanotrophs, indicating that MMO is a similar catalyst to AMO for the purpose of methane oxidation. Furthermore, methanotrophic bacteria share many similarities to NH
3 oxidizers such as Nitrosomonas. The inhibitor profile of particulate forms of MMO (pMMO) shows similarity to the profile of AMO, leading to similarity in properties between MMO in methanotrophs and AMO in autotrophs.
Nitrification inhibitors are also of interest from an environmental standpoint because of the production of nitrates and nitrous oxide from the process of nitrification. Nitrous oxide (N2O), although its atmospheric concentration is much lower than that of CO2, has a global warming potential of about 300 times greater than carbon dioxide and contributes 6% of planetary warming due to greenhouse gases. This compound is also notable for catalyzing the breakup of ozone in the stratosphere. Nitrates, a toxic compound for wildlife and livestock and a product of nitrification, are also of concern.
Soil, consisting of polyanionic clays and silicates, generally has a net anionic charge. Consequently, ammonium (NH4+) binds tightly to the soil but nitrate ions (NO3−) do not. Because nitrate is more mobile, it leaches into groundwater supplies through agricultural runoff. Wildlife such as amphibians, freshwater fish, and insects are sensitive to nitrate levels, and have been known to cause death and developmental anomalies in affected species. In addition, because they easily leach into groundwater, contributing to eutrophication, a process in which large algal blooms reduce oxygen levels in bodies of water and lead to death in oxygen-consuming creatures due to anoxia. Nitrification is also thought to contribute to the formation of photochemical smog, ground level ozone, acid rain, changes in species diversity, and other undesirable processes. In addition, nitrification inhibitors have also been shown to suppress the oxidation of methane (CH4), a potent greenhouse gas, to CO2. Both nitrapyrin and acetylene are shown to be especially strong suppressors of both processes, although the modes of action distinguishing them are unclear.
Urine patches in cattle pastures generate large concentrations of the greenhouse gas nitrous oxide through nitrification and denitrification processes in urine-contaminated soils. Over the past few decades, the cattle population has increased more rapidly than the human population. Between the years 2000 and 2050, the cattle population is expected to increase from 1.5 billion to 2.6 billion. When large populations of cattle are packed into pastures, excessive amounts of urine soak into soils. This increases the rate at which nitrification and denitrification occur and produce nitrous oxide. Currently, nitrous oxide is one of the single most important ozone-depleting emissions and is expected to remain the largest throughout the 21st century.Comammox
Comammox (COMplete AMMonia OXidiser) is the name for an organism that can first convert ammonia into nitrite and then into nitrate by a process called nitrification. These two processes are commonly carried out by separate groups of microorganisms. However, complete conversion of ammonia into nitrate by a single microorganism was predicted in 2006. Almost ten years later the presence of such organisms was discovered within the nitrospira and the nitrogen cycle had to be updated. The genomes revealed the presence of genes necessary for ammonia oxidation (e.g. amoA gene and hao cluster). Nearly two years after the discovery of comammox organisms, Nitrospira inopinata was the first complete nitrifier to be isolated in pure culture. Kinetic and physiological analysis of Nitrospira inopinata demonstrated that this complete nitrifier has a high affinity for ammonia, slow growth rate, low maximum rate of ammonia oxidation, and high yield.Constructed wetland
A constructed wetland (CW) is an artificial wetland to treat municipal or industrial wastewater, greywater or stormwater runoff. It may also be designed for land reclamation after mining, or as a mitigation step for natural areas lost to land development.
Constructed wetlands are engineered systems that use natural functions vegetation, soil, and organisms to treat wastewater. Depending on the type of wastewater the design of the constructed wetland has to be adjusted accordingly. Constructed wetlands have been used to treat both centralized and on-site wastewater. Primary treatment is recommended when there is a large amount of suspended solids or soluble organic matter (measured as BOD and COD).Similarly to natural wetlands, constructed wetlands also act as a biofilter and/or can remove a range of pollutants (such as organic matter, nutrients, pathogens, heavy metals) from the water. Constructed wetlands are a sanitation technology that have not been designed specifically for pathogen removal, but instead, have been designed to remove other water quality constituents such as suspended solids, organic matter and nutrients (nitrogen and phosphorus). All types of pathogens (i.e., bacteria, viruses, protozoan and helminths) are expected to be removed to some extent in a constructed wetland. Subsurface wetland provide greater pathogen removal than surface wetlands.There are two main types of constructed wetlands: subsurface flow and surface flow constructed wetlands. The planted vegetation plays an important role in contaminant removal. The filter bed, consisting usually of sand and gravel, has an equally important role to play. Some constructed wetlands may also serve as a habitat for native and migratory wildlife, although that is not their main purpose. Subsurface flow constructed wetlands are designed to have either horizontal flow or vertical flow of water through the gravel and sand bed. Vertical flow systems have a smaller space requirement than horizontal flow systems.F-ratio
In oceanic biogeochemistry, the f-ratio is the fraction of total primary production fuelled by nitrate (as opposed to that fuelled by other nitrogen compounds such as ammonium). The ratio was originally defined by Richard Eppley and Bruce Peterson in one of the first papers estimating global oceanic production. This fraction was originally believed significant because it appeared to directly relate to the sinking (export) flux of organic marine snow from the surface ocean by the biological pump. However, this interpretation relied on the assumption of a strong depth-partitioning of a parallel process, nitrification, that more recent measurements has questioned.Human impact on the nitrogen cycle
Human impact on the nitrogen cycle is diverse. Agricultural and industrial nitrogen (N) inputs to the environment currently exceed inputs from natural N fixation. As a consequence of anthropogenic inputs, the global nitrogen cycle (Fig. 1) has been significantly altered over the past century. Global atmospheric nitrous oxide (N2O) mole fractions have increased from a pre-industrial value of ~270 nmol/mol to ~319 nmol/mol in 2005. Human activities account for over one-third of N2O emissions, most of which are due to the agricultural sector. This article is intended to give a brief review of the history of anthropogenic N inputs, and reported impacts of nitrogen inputs on selected terrestrial and aquatic ecosystems.Hydroxylamine
Hydroxylamine is an inorganic compound with the formula NH2OH. The pure material is a white, unstable crystalline, hygroscopic compound. However, hydroxylamine is almost always provided and used as an aqueous solution. It is used to prepare oximes, an important functional group. It is also an intermediate in biological nitrification. In biological nitrification, the oxidation of NH3 to hydroxylamine is mediated by the enzyme ammonia monooxygenase (AMO). Hydroxylamine oxidoreductase (HAO) further oxidizes hydroxylamine to nitrite.Karanjin
Karanjin is a furanoflavonol, a type of flavonoid. It is obtained from the seeds of the karanja tree (Millettia pinnata or Pongamia glabra Vent.), a tree growing wild in south India. Karanjin is an acaricide and insecticide. Karanjin is reported to have nitrification inhibitory properties.Microbial metabolism
Microbial metabolism is the means by which a microbe obtains the energy and nutrients (e.g. carbon) it needs to live and reproduce. Microbes use many different types of metabolic strategies and species can often be differentiated from each other based on metabolic characteristics. The specific metabolic properties of a microbe are the major factors in determining that microbe's ecological niche, and often allow for that microbe to be useful in industrial processes or responsible for biogeochemical cycles.Nitrapyrin
Nitrapyrin is an organic compound with the formula ClC5H3NCCl3. It is a widely used nitrification inhibitor in agriculture as well as a soil bactericide and has been in use since 1974. Nitrapyrin was put up for review by the EPA and deemed safe for use in 2005. Since nitrapyrin is an effective nitrification inhibitor to the bacteria nitrosomonas it has been shown to drastically the reduce NO2 emissions of soil. Nitrapyrin is a white crystalline solid with a sweet odor and is often mixed with anhydrous ammonia for application.Nitrifying bacteria
Nitrifying bacteria are chemolithotrophic organisms that include species of the genera Nitrosomonas, Nitrosococcus, Nitrobacter and Nitrococcus. These bacteria get their energy by the oxidation of inorganic nitrogen compounds. Types include ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB). Many species of nitrifying bacteria have complex internal membrane systems that are the location for key enzymes in nitrification: ammonia monooxygenase (which oxidizes ammonia to hydroxylamine), hydroxylamine oxidoreductase (which oxidizes hydroxylamine to nitric oxide - which is oxidized to nitrite by a currently unidentified enzyme), and nitrite oxidoreductase (which oxidizes nitrite to nitrate).Nitrobacter
Nitrobacter is a genus comprising rod-shaped, gram-negative, and chemoautotrophic bacteria. The name Nitrobacter derives from the Latin neuter gender noun nitrum, nitri, alkalis; the Ancient Greek noun βακτηρία, βακτηρίᾱς, rod. They are non-motile and reproduce via budding or binary fission. Nitrobacter cells are obligate aerobes and have a doubling time of about 13 hours.Nitrobacter play an important role in the nitrogen cycle by oxidizing nitrite into nitrate in soil and marine systems. Unlike plants, where electron transfer in photosynthesis provides the energy for carbon fixation, Nitrobacter uses energy from the oxidation of nitrite ions, NO2−, into nitrate ions, NO3−, to fulfill their energy needs. Nitrobacter fix carbon dioxide via the Calvin cycle for their carbon requirements. Nitrobacter belongs to the α-subclass of the Proteobacteria.Nitrogen cycle
The nitrogen cycle is the biogeochemical cycle by which nitrogen is converted into multiple chemical forms as it circulates among atmosphere, terrestrial, and marine ecosystems. The conversion of nitrogen can be carried out through both biological and physical processes. Important processes in the nitrogen cycle include fixation, ammonification, nitrification, and denitrification. The majority of Earth's atmosphere (78%) is atmosphere nitrogen, making it the largest source of nitrogen. However, atmospheric nitrogen has limited availability 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. Human modification of global nitrogen cycle can negatively affect the natural environment system and also human health.Nitrosomonadales
The Nitrosomonadales are an order of the class Betaproteobacteria in the phylum "Proteobacteria". Like all members of their class, they are Gram-negative.
The order is divided into six families:Nitrosomonadaceae (type family) comprises the genera Nitrosomonas (type genus), Nitrosolobus and Nitrosospira. Methylophilaceae comprises the genera Methylophilus (type genus), Methylobacillus and Methylovorus. Spirillaceae comprises the genus Spirillum (type genus) Thiobacillaceae comprises the genera Thiobacillus (type genus), Annwoodia, Sulfuritortus. Gallionellaceae comprises the genera Gallionella (type genus), Ferriphaselus, Sulfuriferula, Sulfurirhabdus and Sulfuricella. Sterolibacteraceae comprises the genera Sterolibacterium, Sulfurisoma, Denitratisoma, Sulfuritalea, Georgfuchsia, Sulfurisoma and Methyloversatilis.Members of the genus Nitrosomonas oxidize ammonium ions into nitrite, - a process called nitrification - and are important in the nitrogen cycle. Other autotrophic genera such as Thiobacillus and Annwoodia oxidize reduced inorganic sulfur ions such as thiosulfate and sulfide into sulfate and have key roles in the sulfur cycle. Methylotrophs such as Methylophilus oxidize compounds such as methanol into carbon dioxide and are key to the carbon cycle. Gallionella and Ferriphaselus oxidise ferric iron (Fe3+) ions into ferric hydroxide (Fe(OH)3) during autotrophic growth, and thus have roles in the carbon cycle and the iron cycle. As such, the Nitrosomonadales are critical to biogeochemical cycling of the elements and many species have key roles in principal biochemical processes.Nitrospira
Nitrospira (from Latin: nitro, meaning "nitrate" and Greek: spira, meaning "spiral") translate into “a nitrate spiral” is a genus of bacteria within the monophyletic clade of Nitrospirae phylum. The first member of this genus was described 1986 by Watson et al. isolated from the Gulf of Maine. The bacterium was named Nitrospira marina. Populations were initially thought to be limited to marine ecosystems, but it was later discovered to be well-suited for numerous habitats, including activated sludge of wastewater treatment systems, natural biological marine settings (such as the Seine River in France and beaches in Cape Cod), water circulation biofilters in aquarium tanks, terrestrial systems, fresh and salt water ecosystems, and hot springs.
Nitrospira is a ubiquitous bacterium that plays a role in the nitrogen cycle by performing nitrite oxidation in the second step of nitrification. Nitrospira live in a wide array of environments including but not limited to, drinking water systems, waste treatment plants, rice paddies, forest soils, geothermal springs, and sponge tissue. Despite being abundant in many natural and engineered ecosystems Nitrospira are difficult to culture, so most knowledge of them is from molecular and genomic data. However, due to their difficulty to be cultivated in laboratory settings, the entire genome was only sequenced in one species, Nitrospira defluvii. In addition, Nitrospira bacteria's 16s rRNA sequences are too dissimilar to use for PCR primers, thus some members go unnoticed. In addition, members of Nitrospira with the capabilities to perform complete nitrification (Comammox bacteria) has also been discovered.Organic hydroponics
Organic hydroponics is a hydroponics culture system which is managed based on organic agriculture concepts.
Most studies have focused on use of organic fertilizer. Conventional hydroponics have a difficult time using organic compounds as fertilizer.In this method of organic hydroponics, organic fertilizer is degraded into inorganic nutrients by microorganisms in the hydroponic solution via ammonification and nitrification.
The microorganisms are cultured with a method of multiple parallel mineralization. The culture solution can be used as the hydroponic solution. Practical method of organic hydroponics is developed in National Agriculture and Food Research Organization (NARO), in Japan, in 2005.Pool frog
The pool frog (Pelophylax lessonae) is a European frog. It is one of only four amphibian species recognized by the UK government as protected under its Biodiversity Action Plan. The reasons for declining populations are decreased pond habitat from human encroachment and also air pollution leading to over-nitrification of pond waters. Its specific name was chosen by the Italian herpetologist Lorenzo Camerano in order to honour his master Michele Lessona.Potassium azide
Potassium azide is the inorganic compound having the formula KN3. It is a white, water-soluble salt. It is used as a reagent in the laboratory.
It has been found to act as a nitrification inhibitor in soil.Simultaneous nitrification–denitrification
Simultaneous nitrification–denitrification (SNdN) is a wastewater treatment process. Microbial simultaneous nitrification-denitrification is the conversion of the ammonium ion to nitrogen gas in a single bioreactor. The process is dependent on floc characteristics, reaction kinetics, mass loading of readily biodegradable chemical oxygen demand, rbCOD, and the dissolved oxygen, DO, concentrationSoil biology
Soil biology is the study of microbial and faunal activity and ecology in soil.
Soil life, soil biota, soil fauna, or edaphon is a collective term that encompasses all organisms that spend a significant portion of their life cycle within a soil profile, or at the soil-litter interface.
These organisms include earthworms, nematodes, protozoa, fungi, bacteria, different arthropods, as well as some reptiles (such as snakes), and species of burrowing mammals like gophers, moles and prairie dogs. Soil biology plays a vital role in determining many soil characteristics. The decomposition of organic matter by soil organisms has an immense influence on soil fertility, plant growth, soil structure, and carbon storage. As a relatively new science, much remains unknown about soil biology and its effect on soil ecosystems.