nitrification

The process of nitrification begins with the first stage of ammonia oxidation, where ammonia (NH₃) or ammonium (NH₄⁺) get converted into nitrite (NO₂⁻). This first stage is sometimes known as nitritation. It is performed by two groups of organisms, ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA{{cite journal | vauthors = Hatzenpichler R | title = Diversity, physiology, and niche differentiation of ammonia-oxidizing archaea | journal = Applied and Environmental Microbiology | volume = 78 | issue = 21 | pages = 7501–10 | date = November 2012 | pmid = 22923400 | pmc = 3485721 | doi = 10.1128/aem.01960-12 | bibcode = 2012ApEnM..78.7501H }}).

Ammonia-Oxidizing Bacteria (AOB) are typically Gram-negative bacteria and belong to Betaproteobacteria and Gammaproteobacteria{{cite journal | vauthors = Purkhold U, Pommerening-Röser A, Juretschko S, Schmid MC, Koops HP, Wagner M | title = Phylogeny of all recognized species of ammonia oxidizers based on comparative 16S rRNA and amoA sequence analysis: implications for molecular diversity surveys | journal = Applied and Environmental Microbiology | volume = 66 | issue = 12 | pages = 5368–82 | date = December 2000 | pmid = 11097916 | pmc = 92470 | doi = 10.1128/aem.66.12.5368-5382.2000 | bibcode = 2000ApEnM..66.5368P }} including the commonly studied genera Nitrosomonas and Nitrococcus. They are known for their ability to utilize ammonia as an energy source and are prevalent in a wide range of environments, such as soils, aquatic systems, and wastewater treatment plants.

AOB possess enzymes called ammonia monooxygenases (AMOs), which are responsible for catalyzing the conversion of ammonia to hydroxylamine (NH₂OH), a crucial intermediate in the process of nitrification.{{Cite journal |last1=Wright |first1=Chloë L. |last2=Schatteman |first2=Arne |last3=Crombie |first3=Andrew T. |last4=Murrell |first4=J. Colin |last5=Lehtovirta-Morley |first5=Laura E. |date=2020-04-17 |title=Inhibition of Ammonia Monooxygenase from Ammonia-Oxidizing Archaea by Linear and Aromatic Alkynes |url=http://dx.doi.org/10.1128/aem.02388-19 |journal=Applied and Environmental Microbiology |volume=86 |issue=9 |pages=e02388-19 |doi=10.1128/aem.02388-19 |pmid=32086308 |issn=0099-2240|pmc=7170481 |bibcode=2020ApEnM..86E2388W }} This enzymatic activity is sensitive to environmental factors, such as pH, temperature, and oxygen availability.

AOB play a vital role in soil nitrification, making them key players in nutrient cycling. They contribute to the transformation of ammonia derived from organic matter decomposition or fertilizers into nitrite, which subsequently serves as a substrate for nitrite-oxidizing bacteria (NOB).

Prior to the discovery of archaea capable of ammonia oxidation, ammonia-oxidizing bacteria (AOB) were considered the only organisms capable of ammonia oxidation. Since their discovery in 2005,{{cite journal | vauthors = Treusch AH, Leininger S, Kletzin A, Schuster SC, Klenk HP, Schleper C | title = Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling | journal = Environmental Microbiology | volume = 7 | issue = 12 | pages = 1985–95 | date = December 2005 | pmid = 16309395 | doi = 10.1111/j.1462-2920.2005.00906.x | bibcode = 2005EnvMi...7.1985T }} two isolates of AOAs have been cultivated: Nitrosopumilus maritimus{{cite journal | vauthors = Könneke M, Bernhard AE, de la Torre JR, Walker CB, Waterbury JB, Stahl DA | title = Isolation of an autotrophic ammonia-oxidizing marine archaeon | journal = Nature | volume = 437 | issue = 7058 | pages = 543–6 | date = September 2005 | pmid = 16177789 | doi = 10.1038/nature03911 | bibcode = 2005Natur.437..543K | s2cid = 4340386 }} and Nitrososphaera viennensis.{{cite journal | vauthors = Tourna M, Stieglmeier M, Spang A, Könneke M, Schintlmeister A, Urich T, Engel M, Schloter M, Wagner M, Richter A, Schleper C | display-authors = 6 | title = Nitrososphaera viennensis, an ammonia oxidizing archaeon from soil | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 108 | issue = 20 | pages = 8420–5 | date = May 2011 | pmid = 21525411 | pmc = 3100973 | doi = 10.1073/pnas.1013488108 | bibcode = 2011PNAS..108.8420T | doi-access = free }} When comparing AOB and AOA, AOA dominate in both soils and marine environments,{{cite journal | vauthors = Karner MB, DeLong EF, Karl DM | title = Archaeal dominance in the mesopelagic zone of the Pacific Ocean | journal = Nature | volume = 409 | issue = 6819 | pages = 507–10 | date = January 2001 | pmid = 11206545 | doi = 10.1038/35054051 | bibcode = 2001Natur.409..507K | s2cid = 6789859 }}{{cite journal | vauthors = Wuchter C, Abbas B, Coolen MJ, Herfort L, van Bleijswijk J, Timmers P, Strous M, Teira E, Herndl GJ, Middelburg JJ, Schouten S, Sinninghe Damsté JS | display-authors = 6 | title = Archaeal nitrification in the ocean | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 103 | issue = 33 | pages = 12317–22 | date = August 2006 | pmid = 16894176 | pmc = 1533803 | doi = 10.1073/pnas.0600756103 | bibcode = 2006PNAS..10312317W | doi-access = free }}{{cite journal | vauthors = Leininger S, Urich T, Schloter M, Schwark L, Qi J, Nicol GW, Prosser JI, Schuster SC, Schleper C | display-authors = 6 | title = Archaea predominate among ammonia-oxidizing prokaryotes in soils | journal = Nature | volume = 442 | issue = 7104 | pages = 806–9 | date = August 2006 | pmid = 16915287 | doi = 10.1038/nature04983 | url = http://www.abdn.ac.uk/staffpages/uploads/mbi010/Nature%20442,%20806-809.pdf | s2cid = 4380804 | bibcode = 2006Natur.442..806L | author-link7 = James I. Prosser | access-date = 2016-05-18 | archive-date = 2016-06-11 | archive-url = https://web.archive.org/web/20160611030331/http://www.abdn.ac.uk/staffpages/uploads/mbi010/Nature%20442,%20806-809.pdf | url-status = live }}{{cite journal | vauthors = Daebeler A, Abell GC, Bodelier PL, Bodrossy L, Frampton DM, Hefting MM, Laanbroek HJ | title = Archaeal dominated ammonia-oxidizing communities in Icelandic grassland soils are moderately affected by long-term N fertilization and geothermal heating | language = English | journal = Frontiers in Microbiology | volume = 3 | pages = 352 | date = 2012 | pmid = 23060870 | pmc = 3463987 | doi = 10.3389/fmicb.2012.00352 | doi-access = free }} suggesting that Nitrososphaerota (formerly Thaumarchaeota) may be greater contributors to ammonia oxidation in these environments.

Crenarchaeol, which is generally thought to be produced exclusively by AOA (specifically Nitrososphaerota), has been proposed as a biomarker for AOA and ammonia oxidation. Crenarchaeol abundance has been found to track with seasonal blooms of AOA, suggesting that it may be appropriate to use crenarchaeol abundances as a proxy for AOA populations{{Cite journal|last1=Pitcher|first1=Angela|last2=Wuchter|first2=Cornelia|last3=Siedenberg|first3=Kathi|last4=Schouten|first4=Stefan|last5=Sinninghe Damsté|first5=Jaap S.|date=2011|title=Crenarchaeol tracks winter blooms of ammonia-oxidizing Thaumarchaeota in the coastal North Sea|journal=Limnology and Oceanography|volume=56|issue=6|pages=2308–2318|doi=10.4319/lo.2011.56.6.2308|issn=0024-3590|bibcode=2011LimOc..56.2308P|url=http://www.vliz.be/imisdocs/publications/49/256149.pdf|doi-access=free|access-date=2022-08-27|archive-date=2023-05-22|archive-url=https://web.archive.org/web/20230522172309/https://www.vliz.be/imisdocs/publications/49/256149.pdf|url-status=live}} and thus ammonia oxidation more broadly. However the discovery of Nitrososphaerota that are not obligate ammonia-oxidizers{{cite journal|vauthors=Mussmann M, Brito I, Pitcher A, Sinninghe Damsté JS, Hatzenpichler R, Richter A, Nielsen JL, Nielsen PH, Müller A, Daims H, Wagner M, Head IM|date=October 2011|title=Thaumarchaeotes abundant in refinery nitrifying sludges express amoA but are not obligate autotrophic ammonia oxidizers|journal=Proceedings of the National Academy of Sciences of the United States of America|volume=108|issue=40|pages=16771–6|bibcode=2011PNAS..10816771M|doi=10.1073/pnas.1106427108|pmc=3189051|pmid=21930919|doi-access=free }} complicates this conclusion,{{cite journal | vauthors = Rush D, Sinninghe Damsté JS | title = Lipids as paleomarkers to constrain the marine nitrogen cycle | journal = Environmental Microbiology | volume = 19 | issue = 6 | pages = 2119–2132 | date = June 2017 | pmid = 28142226 | pmc = 5516240 | doi = 10.1111/1462-2920.13682 | bibcode = 2017EnvMi..19.2119R }} as does one study that suggests that crenarchaeol may be produced by Marine Group II Euryarchaeota.{{cite journal | vauthors = Lincoln SA, Wai B, Eppley JM, Church MJ, Summons RE, DeLong EF | title = Planktonic Euryarchaeota are a significant source of archaeal tetraether lipids in the ocean | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 111 | issue = 27 | pages = 9858–63 | date = July 2014 | pmid = 24946804 | pmc = 4103328 | doi = 10.1073/pnas.1409439111 | bibcode = 2014PNAS..111.9858L | doi-access = free }}

The second step of nitrification is the oxidation of nitrite into nitrate. This process is sometimes known as nitratation. Nitrite oxidation is conducted by nitrite-oxidizing bacteria (NOB) from the taxa Nitrospirota,{{cite journal | vauthors = Daims H, Nielsen JL, Nielsen PH, Schleifer KH, Wagner M | title = In situ characterization of Nitrospira-like nitrite-oxidizing bacteria active in wastewater treatment plants | journal = Applied and Environmental Microbiology | volume = 67 | issue = 11 | pages = 5273–84 | date = November 2001 | pmid = 11679356 | pmc = 93301 | doi = 10.1128/AEM.67.11.5273-5284.2001 | bibcode = 2001ApEnM..67.5273D | url = }} Nitrospinota,{{cite journal | vauthors = Beman JM, Leilei Shih J, Popp BN | title = Nitrite oxidation in the upper water column and oxygen minimum zone of the eastern tropical North Pacific Ocean | journal = The ISME Journal | volume = 7 | issue = 11 | pages = 2192–205 | date = November 2013 | pmid = 23804152 | pmc = 3806268 | doi = 10.1038/ismej.2013.96 | bibcode = 2013ISMEJ...7.2192B }} Pseudomonadota{{cite journal | vauthors = Poly F, Wertz S, Brothier E, Degrange V | title = First exploration of Nitrobacter diversity in soils by a PCR cloning-sequencing approach targeting functional gene nxrA | journal = FEMS Microbiology Ecology | volume = 63 | issue = 1 | pages = 132–40 | date = January 2008 | pmid = 18031541 | doi = 10.1111/j.1574-6941.2007.00404.x | doi-access = | bibcode = 2008FEMME..63..132P }} and Chloroflexota.{{cite journal | vauthors = Spieck E, Spohn M, Wendt K, Bock E, Shively J, Frank J, Indenbirken D, Alawi M, Lücker S, Hüpeden J | display-authors = 6 | title = Extremophilic nitrite-oxidizing Chloroflexi from Yellowstone hot springs | journal = The ISME Journal | volume = 14 | issue = 2 | pages = 364–379 | date = February 2020 | pmid = 31624340 | pmc = 6976673 | doi = 10.1038/s41396-019-0530-9 | bibcode = 2020ISMEJ..14..364S }} NOB are typically present in soil, geothermal springs, freshwater and marine ecosystems.

{{main|Comammox}}

Ammonia oxidation to nitrate in a single step within one organism was predicted in 2006{{cite journal | vauthors = Costa E, Pérez J, Kreft JU | title = Why is metabolic labour divided in nitrification? | journal = Trends in Microbiology | volume = 14 | issue = 5 | pages = 213–9 | date = May 2006 | pmid = 16621570 | doi = 10.1016/j.tim.2006.03.006 | url = https://linkinghub.elsevier.com/retrieve/pii/S0966842X06000758 | access-date = 2021-01-21 | archive-date = 2020-10-19 | archive-url = https://web.archive.org/web/20201019214506/https://linkinghub.elsevier.com/retrieve/pii/S0966842X06000758 | url-status = live }} and discovered in 2015 in the species Nitrospira inopinata. A pure culture of the organism was obtained in 2017,{{cite journal | vauthors = Kits KD, Sedlacek CJ, Lebedeva EV, Han P, Bulaev A, Pjevac P, Daebeler A, Romano S, Albertsen M, Stein LY, Daims H, Wagner M | display-authors = 6 | title = Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle | journal = Nature | volume = 549 | issue = 7671 | pages = 269–272 | date = September 2017 | pmid = 28847001 | pmc = 5600814 | doi = 10.1038/nature23679 | bibcode = 2017Natur.549..269K | url = }} representing a revolution in our understanding of the nitrification process.

The idea that oxidation of ammonia to nitrate is in fact a biological process was first given by Louis Pasteur in 1862.{{Cite journal| vauthors = Pasteur L |date=1862|title=Etudes sur les mycoderme|journal=C. R. Acad. Sci.|volume=54|pages=265–270}} Later in 1875, Alexander Müller, while conducting a quality assessment of water from wells in Berlin, noted that ammonium was stable in sterilized solutions but nitrified in natural waters. A. Müller put forward, that nitrification is thus performed by microorganisms.{{Cite journal| vauthors = Müller A |date=1875|title=Ammoniakgehalt des Spree- und Wasserleitungs wassers in Berlin|journal=Fortsetzung der Vorarbeiten zu einer zukünftigen Wasser-Versorgung der Stadt Berlin ausgeführt in den Jahren 1868 und 1869.|pages=121–123}} In 1877, Jean-Jacques Schloesing and Achille Müntz, two French agricultural chemists working in Paris, proved that nitrification is indeed microbially mediated process by the experiments with liquid sewage and artificial soil matrix (sterilized sand with powdered chalk).{{Cite journal| vauthors = Schloesing T, Muntz A |date=1877|title=Sur la nitrification pas les ferments organisés|journal=C. R. Acad. Sci.|volume=84|pages=301–303}} Their findings were confirmed soon (in 1878) by Robert Warington who was investigating nitrification ability of garden soil at the Rothamsted experimental station in Harpenden in England.{{Cite journal| vauthors = Warington R |date=1878|title=IV.—On nitrification|url=http://xlink.rsc.org/?DOI=CT8783300044|journal=J. Chem. Soc., Trans.|language=en|volume=33|pages=44–51|doi=10.1039/CT8783300044|issn=0368-1645}} R. Warington made also the first observation that nitrification is a two-step process in 1879{{Cite journal|vauthors=Warington R|date=1879|title=XLIX.—On nitrification. (Part II.)|url=http://xlink.rsc.org/?DOI=CT8793500429|journal=J. Chem. Soc., Trans.|language=en|volume=35|pages=429–456|doi=10.1039/CT8793500429|issn=0368-1645|access-date=2021-03-12|archive-date=2021-06-12|archive-url=https://web.archive.org/web/20210612114744/https://pubs.rsc.org/en/content/articlelanding/1879/CT/CT8793500429|url-status=live}} which was confirmed by John Munro in 1886.{{Cite journal| vauthors = Munro JH |date=1886|title=LIX.—The formation and destruction of nitrates and nitrates in artificial solutions and in river and well waters|url=http://xlink.rsc.org/?DOI=CT8864900632|journal=J. Chem. Soc., Trans.|language=en|volume=49|pages=632–681|doi=10.1039/CT8864900632|issn=0368-1645}} Although at that time, it was believed that two-step nitrification is separated into distinct life phases or character traits of a single microorganism.

The first pure nitrifier (ammonia-oxidizing) was most probably isolated in 1890 by Percy Frankland and Grace Frankland, two English scientists from Scotland.{{Cite journal|date=1890-12-31|title=V. The nitrifying process and its specific ferment.—Part I|journal=Philosophical Transactions of the Royal Society of London B|language=en|volume=181|pages=107–128|doi=10.1098/rstb.1890.0005|issn=0264-3839|doi-access=free}} Before that, Warington, Sergei Winogradsky{{Cite journal| vauthors = Winogradsky S |date=1890|title=Sur les organisms de la nitrification|journal=Ann. Inst. Pasteur|volume=4|pages=215–231}} and the Franklands were only able to enrich cultures of nitrifiers. Frankland and Frankland succeeded with a system of serial dilutions with very low inoculum and long cultivation times counting in years. Sergei Winogradsky claimed pure culture isolation in the same year (1890), but his culture was still co-culture of ammonia- and nitrite-oxidizing bacteria.{{cite journal | vauthors = Sedlacek CJ | title = It Takes a Village: Discovering and Isolating the Nitrifiers | journal = Frontiers in Microbiology | volume = 11 | pages = 1900 | date = 2020-08-11 | pmid = 32849473 | pmc = 7431685 | doi = 10.3389/fmicb.2020.01900 | doi-access = free }} S. Winogradsky succeeded just one year later in 1891.{{Cite journal| vauthors = Winogradsky S |date=1891|title=Sur les organisms de la nitrification|url=|journal=Ann. Inst. Pasteur|volume=5|pages=92–100 }}

In fact, during the serial dilutions ammonia-oxidizers and nitrite-oxidizers were unknowingly separated resulting in pure culture with ammonia-oxidation ability only. Thus Frankland and Frankland observed that these pure cultures lose ability to perform both steps. Loss of nitrite oxidation ability was observed already by R. Warington. Cultivation of pure nitrite oxidizer happened later during 20th century, however it is not possible to be certain which cultures were without contaminants as all theoretically pure strains share same trait (nitrite consumption, nitrate production).

{{biogeochemical cycle sidebar|nutrient}}

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.{{cite journal | vauthors = Marsh KL, Sims GK, Mulvaney RL | year = 2005 | title = Availability of urea to autotrophic ammonia-oxidizing bacteria as related to the fate of ¹⁴C- and ¹⁵N-labeled urea added to soil | journal = Biol. Fert. Soil. | volume = 42 | issue = 2| pages = 137–145 | doi=10.1007/s00374-005-0004-2| bibcode = 2005BioFS..42..137M | s2cid = 6245255 }}

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.{{cite journal | vauthors = Zhang Y, Love N, Edwards M | year = 2009 | title = Nitrification in Drinking Water Systems | journal = Critical Reviews in Environmental Science and Technology | volume = 39 | issue = 3| pages = 153–208 | doi = 10.1080/10643380701631739 | bibcode = 2009CREST..39..153Z | s2cid = 96988652 }} The addition of chlorite ion to chloramine-treated water has been shown to control nitrification.{{cite journal|doi=10.1002/j.1551-8833.1999.tb08715.x|title=Using chlorite ion to control nitrification|journal=Journal - American Water Works Association|volume=91|issue=10|pages=52–61|year=1999| vauthors = McGuire MJ, Lieu NI, Pearthree MS |bibcode=1999JAWWA..91j..52M |s2cid=93321500 }}{{cite journal|doi=10.1002/j.1551-8833.2009.tb09970.x|title=Prevention of nitrification using chlorite ion: Results of a demonstration project in Glendale, Calif|journal=Journal - American Water Works Association|volume=101|issue=10|pages=47–59|year=2009| vauthors = McGuire MJ, Wu X, Blute NK, Askenaizer D, Qin G |bibcode=2009JAWWA.101j..47M |s2cid=101973325 }}

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.

In the marine environment, nitrogen is often the limiting nutrient, so the nitrogen cycle in the ocean is of particular interest.{{cite journal |vauthors=Zehr JP, Kudela RM |year=2011 |title=Nitrogen cycle of the open ocean: from genes to ecosystems |journal=Annual Review of Marine Science |volume=3 |pages=197–225 |bibcode=2011ARMS....3..197Z |doi=10.1146/annurev-marine-120709-142819 |pmid=21329204 |s2cid=23018410}}{{cite journal |vauthors=Ward BB |date=November 1996 |title=Nitrification and Denitrification: Probing the Nitrogen Cycle in Aquatic Environments |url=https://www.princeton.edu/nitrogen/publications/pdfs/Ward_1996_Probing.pdf |journal=Microbial Ecology |volume=32 |issue=3 |pages=247–61 |doi=10.1007/BF00183061 |pmid=8849421 |bibcode=1996MicEc..32..247W |s2cid=11550311 |access-date=2018-10-18 |archive-date=2017-10-19 |archive-url=https://web.archive.org/web/20171019062459/https://www.princeton.edu/nitrogen/publications/pdfs/Ward_1996_Probing.pdf |url-status=live }} 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.{{cite journal |vauthors=Hutchins D, Mulholland M, Fu F |year=2009 |title=Nutrient cycles and marine microbes in a CO₂-enriched ocean |url=https://digitalcommons.odu.edu/cgi/viewcontent.cgi?article=1024&context=oeas_fac_pubs |journal=Oceanography |volume=22 |issue=4 |pages=128–145 |doi=10.5670/oceanog.2009.103 |doi-access=free |access-date=2018-10-18 |archive-date=2018-10-18 |archive-url=https://web.archive.org/web/20181018201544/https://digitalcommons.odu.edu/cgi/viewcontent.cgi?article=1024&context=oeas_fac_pubs |url-status=live }}

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. Diverse 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. Subsequent metagenomic studies and cultivation approaches have revealed that some Thermoproteota (formerly Crenarchaeota) possess AMO. Thermoproteota are abundant in the ocean and some species have a 200 times greater affinity for ammonia than AOB, contrasting with the previous belief that AOB are primarily responsible for nitrification in the ocean.{{cite journal |vauthors=Martens-Habbena W, Berube PM, Urakawa H, de la Torre JR, Stahl DA |date=October 2009 |title=Ammonia oxidation kinetics determine niche separation of nitrifying Archaea and Bacteria |journal=Nature |volume=461 |issue=7266 |pages=976–9 |bibcode=2009Natur.461..976M |doi=10.1038/nature08465 |pmid=19794413 |s2cid=1692603}} Furthermore, though nitrification is classically thought to be vertically separated from primary production because the oxidation of nitrate 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{{cite journal |vauthors=Sun X, Kop LF, Lau MC, Frank J, Jayakumar A, Lücker S, Ward BB |date=October 2019 |title=Uncultured Nitrospina-like species are major nitrite oxidizing bacteria in oxygen minimum zones |journal=The ISME Journal |volume=13 |issue=10 |pages=2391–2402 |doi=10.1038/s41396-019-0443-7 |pmc=6776041 |pmid=31118472|bibcode=2019ISMEJ..13.2391S }} and Nitrobacter are known to carry out this step in the ocean.

Nitrification is a process of nitrogen compound oxidation (effectively, loss of electrons from the nitrogen atom to the oxygen atoms), and is catalyzed step-wise by a series of enzymes.

:2NH4+ + 3O2 -> 2NO2- + 4H+ + 2H2O (Nitrosomonas, Comammox)

:2NO2- + O2 -> 2NO3- (Nitrobacter, Nitrospira, Comammox)

OR

:NH3 + O2 -> NO2- + 3H+ + 2e-

:NO2- + H2O -> NO3- + 2H+ + 2e-

In Nitrosomonas europaea, the first step of oxidation (ammonia to hydroxylamine) is carried out by the enzyme ammonia monooxygenase (AMO).

:NH3 + O2 + 2H+ -> NH2OH + H2O

The second step (hydroxylamine to nitrite) is catalyzed by two enzymes. Hydroxylamine oxidoreductase (HAO), converts hydroxylamine to nitric oxide.{{cite journal | vauthors = Caranto JD, Lancaster KM | title = Nitric oxide is an obligate bacterial nitrification intermediate produced by hydroxylamine oxidoreductase | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 114 | issue = 31 | pages = 8217–8222 | date = August 2017 | pmid = 28716929 | pmc = 5547625 | doi = 10.1073/pnas.1704504114 | bibcode = 2017PNAS..114.8217C | doi-access = free }}

:NH2OH -> NO + 3H+ + 3e-

Another currently unknown enzyme converts nitric oxide to nitrite.

The third step (nitrite to nitrate) is completed in a distinct organism.

:{nitrite} + acceptor <=> {nitrate} + reduced\ acceptor

Due to its inherent microbial nature, nitrification in soils is greatly susceptible to soil conditions. In general, soil nitrification will proceed at optimal rates if the conditions for the microbial communities foster healthy microbial growth and activity. Soil conditions that have an effect on nitrification rates include:

• Substrate availability (presence of NH₄⁺)
• Aeration (availability of O₂)
• Soil moisture content (availability of H₂O)
• pH (near neutral)
• Temperature

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.{{cite journal|doi=10.1094/CM-2007-0510-01-RS|title=An Educational Program on the Proper Timing of Fall-applied Nitrogen Fertilizer|url=https://dl.sciencesocieties.org/publications/cm/abstracts/6/1/2007-0510-01-RS?access=0&view=article|journal=Crop Management|volume=6|pages=1–4|year=2007|vauthors=Czapar GF, Payne J, Tate J}}{{Dead link|date=February 2024 |bot=InternetArchiveBot |fix-attempted=yes }} 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.{{cite journal|doi=10.2136/sssaj2003.0937| vauthors = Ferguson R, Lark R, Slater G |title=Approaches to management zone definition for use of nitrification inhibitors|journal= Soil Sci. Soc. Am. J.|year= 2003|volume= 67|issue=3 |pages=937–947|bibcode=2003SSASJ..67..937F }}

The environmental concerns of nitrification also contribute to interest in the use of nitrification inhibitors: the primary product, nitrate, leaches into groundwater, producing toxicity in both humans{{Cite journal |last1=Duvva |first1=Laxman Kumar |last2=Panga |first2=Kiran Kumar |last3=Dhakate |first3=Ratnakar |last4=Himabindu |first4=Vurimindi |date=2021-12-21 |title=Health risk assessment of nitrate and fluoride toxicity in groundwater contamination in the semi-arid area of Medchal, South India |journal=Applied Water Science |volume=12 |issue=1 |doi=10.1007/s13201-021-01557-4 |issn=2190-5487|doi-access=free }} and some 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 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.

:{{chem|NH₃}} + {{chem|AH₂}} + {{chem|O₂}} → {{chem|NH₂OH}} + A + {{chem|H₂O}}

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:

:{{chem|NH₂OH}} + {{chem|H₂O}} → {{chem|NO₂}}⁻ + 5 H⁺ + 4 e⁻

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 sites 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.{{cite journal|doi=10.1007/s003740050518|title=Modes of action of nitrification inhibitors|journal=Biology and Fertility of Soils|volume=29|pages=1–9|year=1999| vauthors = McCarty GW |issue=1 |bibcode=1999BioFS..29....1M |s2cid=38059676}} 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.

File:Nheterocyclicmolecules.png

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.{{cite journal | vauthors = Topp E, Knowles R | title = Effects of Nitrapyrin [2-Chloro-6-(Trichloromethyl) Pyridine] on the Obligate Methanotroph Methylosinus trichosporium OB3b | journal = Applied and Environmental Microbiology | volume = 47 | issue = 2 | pages = 258–62 | date = February 1984 | pmid = 16346465 | pmc = 239655 | doi = 10.1007/BF01576048 | s2cid = 34551923 }} 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).{{cite journal | vauthors = McCarty GW | year = 1998 | title = Modes of action of nitrification inhibitors | journal = Biology and Fertility of Soils | volume = 29 | issue = 1| pages = 1–9 | doi = 10.1007/s003740050518 | bibcode = 1999BioFS..29....1M | s2cid = 38059676 }} 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 nitrapyrin, can also inhibit the oxidation of methane in methanotrophic bacteria.{{Cite journal |last1=Topp |first1=Edward |last2=Knowles |first2=Roger |date=February 1984 |title=Effects of Nitrapyrin [2-Chloro-6-(Trichloromethyl) Pyridine] on the Obligate Methanotroph Methylosinus trichosporium OB3b |journal=Applied and Environmental Microbiology |language=en |volume=47 |issue=2 |pages=258–262 |doi=10.1128/aem.47.2.258-262.1984 |issn=0099-2240 |pmc=239655 |pmid=16346465|bibcode=1984ApEnM..47..258T }} 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 {{NH3}} oxidizers such as Nitrosomonas.{{cite journal | vauthors = Bédard C, Knowles R | title = Physiology, biochemistry, and specific inhibitors of CH4, NH4+, and CO oxidation by methanotrophs and nitrifiers | journal = Microbiological Reviews | volume = 53 | issue = 1 | pages = 68–84 | date = March 1989 | pmid = 2496288 | pmc = 372717 | doi = 10.1128/MMBR.53.1.68-84.1989 }} 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.

File:Nitrification Process Tank.jpg plant]]

Nitrification inhibitors are also of interest from an environmental standpoint because of the production of nitrates and nitrous oxide from the nitrification process. Nitrous oxide (N₂O), although its atmospheric concentration is much lower than that of CO_2, 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.{{cite journal|doi=10.1017/S1466046607070482|title=Environmental Review: The Potential of Nitrification Inhibitors to Manage the Pollution Effect of Nitrogen Fertilizers in Agricultural and Other Soils: A Review|journal=Environmental Practice|volume=9|issue=4|pages=266–279|year=2007| vauthors = Singh SN, Verma A |s2cid=128612680}} 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. Nitrates in groundwater can affect surface water concentrations through direct groundwater-surface water interactions (e.g., gaining stream reaches, springs) or from when it is extracted for surface use. For example, much of the drinking water in the United States comes from groundwater, but most wastewater treatment plants discharge to surface water.

Among wildlife, amphibians (tadpoles) and freshwater fish eggs are most sensitive to elevated nitrate levels and experience growth and developmental damage at levels commonly found in U.S. freshwater bodies (<20 mg/l). In contrast, freshwater invertebrates are more tolerant (~90+mg/l), and adult freshwater fish can tolerate very high levels (800 mg+/l).{{cite journal | vauthors = Rouse JD, Bishop CA, Struger J | title = Nitrogen pollution: an assessment of its threat to amphibian survival | journal = Environmental Health Perspectives | volume = 107 | issue = 10 | pages = 799–803 | date = October 1999 | pmid = 10504145 | pmc = 1566592 | doi = 10.2307/3454576 | jstor = 3454576 }} Nitrate levels also contribute 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 potent suppressors of both processes, although the modes of action distinguishing them are unclear.

• f-ratio
• Haber process
• Nitrifying bacteria
• Nitrogen fixation
• Simultaneous nitrification-denitrification
• Comammox

{{Reflist}}

• {{usurped|1=[https://web.archive.org/web/20081118013245/http://www.fishdoc.co.uk/filtration/nitrification.htm Nitrification at the heart of filtration]}} at fishdoc.co.uk
• [http://www.abdn.ac.uk/~mbi010/nitrification.htm Nitrification] at University of Aberdeen · King's College
• [http://www.lagoonsonline.com/ripple.htm Nitrification Basics for Aerated Lagoon Operators] at lagoonsonline.com

Category:Biochemical reactions

Category:Nitrogen cycle

Category:Soil biology