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{{Short description|Microbially facilitated process}}(File:Nitrogen Cycle.svg|thumb|320px|Nitrogen cycle.)Denitrification is a microbially facilitated process where nitrate (NO3−) is reduced and ultimately produces molecular nitrogen (N2) through a series of intermediate gaseous nitrogen oxide products. Facultative anaerobic bacteria perform denitrification as a type of respiration that 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), nitrous oxide (N2O) finally resulting in the production of dinitrogen (N2) completing the nitrogen cycle. Denitrifying microbes require a very low oxygen concentration of less than 10%, as well as organic C for energy. Since denitrification can remove NO3−, reducing its leaching to groundwater, it can be strategically used to treat sewage or animal residues of high nitrogen content. Denitrification can leak N2O, which is an ozone-depleting substance and a greenhouse gas that can have a considerable influence on global warming.The process is performed primarily by heterotrophic bacteria (such as Paracoccus denitrificans and various pseudomonads),JOURNAL, Carlson, C. A., Ingraham, J. L., 1983, Comparison of denitrification by Pseudomonas stutzeri, Pseudomonas aeruginosa, and Paracoccus denitrificans, Appl. Environ. Microbiol., 45, 4, 1247–1253, 6407395, 242446, 10.1128/AEM.45.4.1247-1253.1983, 1983ApEnM..45.1247C, although autotrophic denitrifiers have also been identified (e.g., Thiobacillus denitrificans).JOURNAL, Studies on Thiobacillus denitrificans, Archiv für Mikrobiologie, 20, 1, 34–62, 10.1007/BF00412265, 13139524, 1954, Baalsrud, K., Baalsrud, Kjellrun S., 22428082, Denitrifiers are represented in all main phylogenetic groups.JOURNAL, Zumft, W G, 1997, Cell biology and molecular basis of denitrification, Microbiology and Molecular Biology Reviews, 61, 4, 533–616, 10.1128/mmbr.61.4.533-616.1997, 9409151, 232623, Generally several species of bacteria are involved in the complete reduction of nitrate to N2, and more than one enzymatic pathway has been identified in the reduction process.Atlas, R.M., Barthas, R. Microbial Ecology: Fundamentals and Applications. 3rd Ed. Benjamin-Cummings Publishing. {{ISBN|0-8053-0653-6}} The denitrification process does not only provide energy to the organism performing nitrate reduction to dinitrogen gas, but also some anaerobic ciliates can use denitrifying endosymbionts to gain energy similar to the use of mitochondria in oxygen respiring organisms.JOURNAL, Graf, Jon S., Schorn, Sina, Kitzinger, Katharina, Ahmerkamp, Soeren, Woehle, Christian, Huettel, Bruno, Schubert, Carsten J., Kuypers, Marcel M. M., Milucka, Jana, Anaerobic endosymbiont generates energy for ciliate host by denitrification, Nature, 3 March 2021, 591, 7850, 445–450, 10.1038/s41586-021-03297-6, 33658719, 7969357, 2021Natur.591..445G, free, Direct reduction from nitrate to ammonium, a process known as dissimilatory nitrate reduction to ammonium or DNRA,JOURNAL, 10.3354/meps237041, 2002MEPS..237...41A, Dissimilatory nitrate reduction to ammonium (DNRA) as a nitrogen link, versus denitrification as a sink in a shallow estuary (Laguna Madre/Baffin Bay, Texas), Marine Ecology Progress Series, 237, 41–50, An, S., Gardner, WS, 2002, free, is also possible for organisms that have the nrf-gene."MEMBERWIDE">FIRST1= MMM FIRST2=HK FIRST3=BTITLE=THE MICROBIAL NITROGEN-CYCLING NETWORK, Nature Reviews Microbiology, 1, 1, 1–14, 2011, 29398704, 10.1038/nrmicro.2018.9hdl-access=free, BOOK, 10.1007/1-4020-3544-6_13, Spanning, R., Delgado, M. and Richardson, D., 2005, It is possible to encounter DNRA when your source of carbon is a fermentable substrate, as glucose, so if you wanna avoid DNRA use a non fermentable substrate, Nitrogen Fixation: Origins, Applications, and Research Progress, 277–342, The Nitrogen Cycle: Denitrification and its Relationship to N2 Fixation, 978-1-4020-3542-5, 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 genus Pseudomonas, Bradyrhizobium japonicum, and Blastobacter denitrificans.JOURNAL, Liu, X., Tiquia, S. M., Holguin, G., Wu, L., Nold, S. C., Devol, A. H., Luo, K., Palumbo, A. V., Tiedje, J. M., Zhou, J., 2003, Molecular Diversity of Denitrifying Genes in Continental Margin Sediments within the Oxygen-Deficient Zone off the Pacific Coast of Mexico, Appl. Environ. Microbiol., 69, 6, 3549–3560, 10.1128/aem.69.6.3549-3560.2003, 12788762, 161474, 2003ApEnM..69.3549L, 10.1.1.328.2951,

Overview

Half reactions

Denitrification generally proceeds through some combination of the following half reactions, with the enzyme catalyzing the reaction in parentheses:

• NO3− + 2 H+ + 2 e− → {{chem|NO|2}}− + H2O (Nitrate reductase)
• {{chem|NO|2}}− + 2 H+ + e− → NO + H2O (Nitrite reductase)
• 2 NO + 2 H+ + 2 e− → {{chem|N|2|O}} + H2O (Nitric-oxide reductase)
• {{chem|N|2|O}} + 2 H+ + 2 e− → {{chem|N|2 }} + H2O (Nitrous-oxide reductase)

The complete process can be expressed as a net balanced redox reaction, where nitrate (NO3−) gets fully reduced to dinitrogen (N2):

• 2 NO3− + 10 e− + 12 H+ → N2 + 6 H2O

Conditions of denitrification

In nature, denitrification can take place in both terrestrial and marine ecosystems.JOURNAL, Seitzinger, S., Harrison, J. A., Bohlke, J. K., Bouwman, A. F., Lowrance, R., Peterson, B., Tobias, C., Drecht, G. V., 2006, Denitrification Across Landscapes and Waterscapes: A Synthesis, Ecological Applications, 16, 6, 2064–2090, 10.1890/1051-0761(2006)016[2064:dalawa]2.0.co;2, 17205890, 1912/4707, free, Typically, denitrification occurs in anoxic environments, where the concentration of dissolved and freely available oxygen is depleted. In these areas, nitrate (NO3−) or nitrite ({{chem|NO|2}}−) can be used as a substitute terminal electron acceptor instead of oxygen (O2), a more energetically favourable electron acceptor. Terminal electron acceptor is a compound that gets reduced in the reaction by receiving electrons. Examples of anoxic environments can include soils,JOURNAL, Scaglia, J., Lensi, R., Chalamet, A., 20602996, 1985, Relationship between photosynthesis and denitrification in planted soil, Plant and Soil, 84, 1, 37–43, 10.1007/BF02197865, 1985PlSoi..84...37S, groundwater,JOURNAL, Korom, Scott F., 1992, Natural Denitrification in the Saturated Zone: A Review, Water Resources Research, 28, 6, 1657–1668, 1992WRR....28.1657K, 10.1029/92WR00252, wetlands, oil reservoirs,JOURNAL, Cornish Shartau, S. L., Yurkiw, M., Lin, S., Grigoryan, A. A., Lambo, A., Park, H. S., Lomans, B. P., Van Der Biezen, E., Jetten, M. S. M., 2010, Ammonium Concentrations in Produced Waters from a Mesothermic Oil Field Subjected to Nitrate Injection Decrease through Formation of Denitrifying Biomass and Anammox Activity, Applied and Environmental Microbiology, 76, 15, 4977–4987, 10.1128/AEM.00596-10, 2916462, 20562276, Voordouw, G., 2010ApEnM..76.4977C, poorly ventilated corners of the ocean and seafloor sediments.Furthermore, denitrification can occur in oxic environments as well. High activity of denitrifiers can be observed in the intertidal zones, where the tidal cycles cause fluctuations of oxygen concentration in sandy coastal sediments.JOURNAL, Merchant, et al, 2017, Denitrifying community in coastal sediments performs aerobic and anaerobic respiration simultaneously, 28463234, 5520038, The ISME Journal, 11, 8, 1799–1812, 10.1038/ismej.2017.51, 2017ISMEJ..11.1799M, For example, the bacterial species Paracoccus denitrificans engages in denitrification under both oxic and anoxic conditions simultaneously. Upon oxygen exposure, the bacteria is able to utilize nitrous oxide reductase, an enzyme that catalyzes the last step of denitrification.JOURNAL, Qu, et al, 2016, Transcriptional and metabolic regulation of denitrification in Paracoccus denitrificans allows low but significant activity of nitrous oxide reductase under oxic conditions, 26568281, Environmental Microbiology, 18, 9, 2951–63, 10.1111/1462-2920.13128, 2016EnvMi..18.2951Q, Aerobic denitrifiers are mainly Gram-negative bacteria in the phylum Proteobacteria. Enzymes NapAB, NirS, NirK and NosZ are located in the periplasm, a wide space bordered by the cytoplasmic and the outer membrane in Gram-negative bacteria.JOURNAL, Ji, Bin, Yang, Kai, Zhu, Lei, Jiang, Yu, Wang, Hongyu, Zhou, Jun, Zhang, Huining, 85744076, 2015, Aerobic denitrification: A review of important advances of the last 30 years, Biotechnology and Bioprocess Engineering, 20, 4, 643–651, 10.1007/s12257-015-0009-0, A variety of environmental factors can influence the rate of denitrification on an ecosystem-wide scale. For example, temperature and pH have been observed to impact denitrification. In bacterial species Pseudomonas mandelii, expression of denitrifying genes was reduced at temperatures below 30 Â°C and a pH below 5, while activity was largely unaffected between a pH of 6-8.JOURNAL, Saleh-Lakha, Saleema, Shannon, Kelly E., Henderson, Sherri L., Goyer, Claudia, Trevors, Jack T., Zebarth, Bernie J., Burton, David L., 2009-06-15, Effect of pH and Temperature on Denitrification Gene Expression and Activity in Pseudomonas mandelii, Applied and Environmental Microbiology, 75, 12, 3903–3911, 10.1128/AEM.00080-09, 0099-2240, 2698340, 19376915, 2009ApEnM..75.3903S, Organic carbon as an electron donor is a common limiting nutrient for denitrification as observed in benthic sediments and wetlands.JOURNAL, Starr, Robert C., Gillham, Robert W., November 1993, Denitrification and Organic Carbon Availability in Two Aquifers,weblink Groundwater, 31, 6, 934–947, 10.1111/j.1745-6584.1993.tb00867.x, 1993GrWat..31..934S, 0017-467X, JOURNAL, Sirivedhin, Tanita, Gray, Kimberly A., February 2006, Factors affecting denitrification rates in experimental wetlands: Field and laboratory studies,weblink Ecological Engineering, 26, 2, 167–181, 10.1016/j.ecoleng.2005.09.001, 2006EcEng..26..167S, 0925-8574, Nitrate and oxygen can also be potential limiting factors for denitrification, although the latter only has an observed limiting effect in wet soils.JOURNAL, Burgin, Amy J., Groffman, Peter M., Lewis, David N., September 2010, Factors Regulating Denitrification in a Riparian Wetland,weblink Soil Science Society of America Journal, 74, 5, 1826–1833, 10.2136/sssaj2009.0463, 2010SSASJ..74.1826B, 0361-5995, Oxygen likely affects denitrification in multiple ways—because most denitrifiers are facultative, oxygen can inhibit rates, but it can also stimulate denitrification by facilitating nitrification and the production of nitrate. In wetlands as well as deserts,JOURNAL, Peterjohn, William T., Schlesinger, William H., November 1991, Factors Controlling Denitrification in a Chihuahuan Desert Ecosystem,weblink Soil Science Society of America Journal, 55, 6, 1694–1701, 10.2136/sssaj1991.03615995005500060032x, 1991SSASJ..55.1694P, 0361-5995, moisture is an environmental limiter on rates of denitrification.Additionally, environmental factors can also influence whether denitrification proceeds to completion, characterized by the complete reduction of NO3- to N2 rather than releasing N2O as an end product. Soil pH and texture are both factors that can moderate denitrification, with higher pH levels driving the reaction more to completion.JOURNAL, Foltz, Mary E., Alesso, Agustín, Zilles, Julie L., 2023, Field soil properties and experimental nutrient additions drive the nitrous oxide ratio in laboratory denitrification experiments: a systematic review, Frontiers in Soil Science, 3, 10.3389/fsoil.2023.1194825, free, 2673-8619, Nutrient composition, particularly the ratio of carbon to nitrogen, is a strong contributor to complete denitrification,JOURNAL, Yang, Xinping, Wang, Shimei, Zhou, Lixiang, January 2012, Effect of carbon source, C/N ratio, nitrate and dissolved oxygen concentration on nitrite and ammonium production from denitrification process by Pseudomonas stutzeri D6,weblink Bioresource Technology, 104, 65–72, 10.1016/j.biortech.2011.10.026, 22074905, 2012BiTec.104...65Y, 0960-8524, with a 2:1 ratio of C:N being able to facilitate full nitrate reduction regardless of temperature or carbon source.JOURNAL, Elefsiniotis, P., Li, D., 2006-02-15, The effect of temperature and carbon source on denitrification using volatile fatty acids,weblink Biochemical Engineering Journal, 28, 2, 148–155, 10.1016/j.bej.2005.10.004, 2006BioEJ..28..148E, 1369-703X, Copper, as a co-factor for nitrite reductase and nitrous-oxide reductase, also promoted complete denitrification when added as a supplement.JOURNAL, Moloantoa, Karabelo M., Khetsha, Zenzile P., Kana, Gueguim E. B., Maleke, Maleke M., Van Heerden, Esta, Castillo, Julio C., Cason, Errol D., 2023, Metagenomic assessment of nitrate-contaminated mine wastewaters and optimization of complete denitrification by indigenous enriched bacteria, Frontiers in Environmental Science, 11, 10.3389/fenvs.2023.1148872, free, 2296-665X, Besides nutrients and terrain, microbial community composition can also affect the ratio of complete denitrification, with prokaryotic phyla Actinomycetota and Thermoproteota being responsible for greater release of N2 than N2O compared to other prokaryotes.JOURNAL, Deveautour, C., Rojas-Pinzon, P.A., Veloso, M., Rambaud, J., Duff, A.M., Wall, D., Carolan, R., Philippot, L., Richards, K.G., O'Flaherty, V., Brennan, F., May 2022, Biotic and abiotic predictors of potential N2O emissions from denitrification in Irish grasslands soils: A national-scale field study,weblink Soil Biology and Biochemistry, 168, 108637, 10.1016/j.soilbio.2022.108637, 0038-0717, free, Denitrification can lead to a condition called isotopic fractionation in the soil environment. The two stable isotopes of nitrogen, 14N and 15N are both found in the sediment profiles. The lighter isotope of nitrogen, 14N, is preferred during denitrification, leaving the heavier nitrogen isotope, 15N, in the residual matter. This selectivity leads to the enrichment of 14N in the biomass compared to 15N.JOURNAL, Dähnke K., Thamdrup B., 2013, Nitrogen isotope dynamics and fractionation during sedimentary denitrification in Boknis Eck, Baltic Sea, Biogeosciences, 10, 5, 3079–3088, Copernicus Publications, 10.5194/bg-10-3079-2013, 2013BGeo...10.3079D, free, Moreover, the relative abundance of 14N can be analyzed to distinguish denitrification apart from other processes in nature.

Use in wastewater treatment

{{Further|Sewage treatment}}Denitrification is commonly used to remove nitrogen from sewage and municipal wastewater. It is also an instrumental process in constructed wetlandsJOURNAL, Bachand, P. A. M., Horne, A. J., 1999, Denitrification in constructed free-water surface wetlands: II. Effects of vegetation and temperature, Ecological Engineering, 14, 1–2, 17–32, 10.1016/s0925-8574(99)00017-8, 1999EcEng..14...17B, and riparian zonesJOURNAL, Martin, T. L., Kaushik, N. K., Trevors, J. T., Whiteley, H. R., 1999, Review: Denitrification in temperate climate riparian zones, Water, Air, and Soil Pollution, 111, 171–186, 10.1023/a:1005015400607, 1999WASP..111..171M, 96384737, for the prevention of groundwater pollution with nitrate resulting from excessive agricultural or residential fertilizer usage.JOURNAL, Mulvaney, R. L., Khan, S. A., Mulvaney, C. S., 18518, 1997, Nitrogen fertilizers promote denitrification, Biology and Fertility of Soils, 24, 2, 211–220, 10.1007/s003740050233, 1997BioFS..24..211M, Wood chip bioreactors have been studied since the 2000s and are effective in removing nitrate from agricultural run offJOURNAL, Ghane, E, Fausey, NR, Brown, LC, Jan 2015, Modeling nitrate removal in a denitrification bed, Water Res., 71C, 294–305, 10.1016/j.watres.2014.10.039, 25638338, 2015WatRe..71..294G, {{subscription required}} and even manure.JOURNAL, Carney KN, Rodgers M, Lawlor, PG, Zhan, X, 2013, Treatment of separated piggery anaerobic digestate liquid using woodchip biofilters, Environ Technology, 34, 5–8, 663–70, 10.1080/09593330.2012.710408, 23837316, 2013EnvTe..34..663C, 10397713, {{subscription required}}Reduction under anoxic conditions can also occur through process called anaerobic ammonium oxidation (anammox):JOURNAL, Dalsgaard, T., Thamdrup, B., Canfield, D. E., 2005, Anaerobic ammonium oxidation (anammox) in the marine environment, Research in Microbiology, 156, 4, 457–464, 10.1016/j.resmic.2005.01.011, 15862442, free, In some wastewater treatment plants, compounds such as methanol, ethanol, acetate, glycerin, or proprietary products are added to the wastewater to provide a carbon and electron source for denitrifying bacteria.JOURNAL, Chen, K.-C., Lin, Y.-F., 1993, The relationship between denitrifying bacteria and methanogenic bacteria in a mixed culture system of acclimated sludges, Water Research, 27, 12, 1749–1759, 10.1016/0043-1354(93)90113-v, 1993WatRe..27.1749C, The microbial ecology of such engineered denitrification processes is determined by the nature of the electron donor and the process operating conditions.JOURNAL, Baytshtok, Vladimir, Lu, Huijie, Park, Hongkeun, Kim, Sungpyo, Yu, Ran, Chandran, Kartik, 2009-04-15, Impact of varying electron donors on the molecular microbial ecology and biokinetics of methylotrophic denitrifying bacteria, Biotechnology and Bioengineering, 102, 6, 1527–1536, 10.1002/bit.22213, 19097144, 6445650, JOURNAL, Lu, Huijie, Chandran, Kartik, Stensel, David, November 2014, Microbial ecology of denitrification in biological wastewater treatment, Water Research, 64, 237–254, 10.1016/j.watres.2014.06.042, 25078442, 2014WatRe..64..237L, Denitrification processes are also used in the treatment of industrial wastewater.JOURNAL, Constantin, H., Fick, M., 1997, Influence of C-sources on the denitrification rate of a high-nitrate concentrated industrial wastewater, Water Research, 31, 3, 583–589, 10.1016/s0043-1354(96)00268-0, 1997WatRe..31..583C, Many denitrifying bioreactor types and designs are available commercially for the industrial applications, including Electro-Biochemical Reactors (EBRs), membrane bioreactors (MBRs), and moving bed bioreactors (MBBRs).Aerobic denitrification, conducted by aerobic denitrifiers, may offer the potential to eliminate the need for separate tanks and reduce sludge yield. There are less stringent alkalinity requirements because alkalinity generated during denitrification can partly compensate for the alkalinity consumption in nitrification.

Non-biological denitrification

A variety of non-biological methods can remove nitrate. These include methods that can destroy nitrogen compounds, such as chemical and electrochemical methods, and those that selectively transfer nitrate to a concentrated waste stream, such as ion exchange or reverse osmosis. Chemical removal of nitrate can occur through advanced oxidation processes, although it may produce hazardous byproducts.JOURNAL, Rayaroth, Manoj P., Aravindakumar, Charuvila T., Shah, Noor S., Boczkaj, Grzegorz, Advanced oxidation processes (AOPs) based wastewater treatment - unexpected nitration side reactions - a serious environmental issue: A review, Chemical Engineering Journal, Elsevier BV, 430, 2022, 1385-8947, 10.1016/j.cej.2021.133002, 133002, free, 2022ChEnJ.43033002R, Electrochemical methods can remove nitrate by via a voltage applied across electrodes, with degradation usually occurring at the cathode. Effective cathode materials include transition metals, post transition metals,JOURNAL, Rajmohan, K. S., Gopinath, M., Chetty, Raghuram, Review on challenges and opportunities in the removal of nitrate from wastewater using electrochemical method, Triveni Enterprises, 37, 2016, 2394-0379, 1519–1528, and semi-conductors like TiO2.JOURNAL, Ji, Yangyuan, Niu, Junfeng, Xu, Dong, Wang, Kaixuan, Brejcha, Jacob, Jeon, Seunghyo, Warsinger, David M, Efficient electrocatalysis for denitrification by using TiO2 nanotube arrays cathode and adding chloride ions, Chemosphere, Elsevier BV, 274, 2021, 0045-6535, 10.1016/j.chemosphere.2021.129706, 129706, 33540319, 2021Chmsp.27429706J, 231818217,weblink Electrochemical methods can often avoid requiring costly chemical additives, but their effectiveness can be constrained by the pH and ions present. Reverse osmosis is highly effective in removing small charged solutes like nitrate, but it may also remove desirable nutrients, create large volumes of wastewater, and require increased pumping pressures. Ion exchange can selectively remove nitrate from water without large waste streams,JOURNAL, Krueger, Gordon M., A method for the removal of nitrates from waterprior to use in infant formula, The Journal of Pediatrics, Elsevier BV, 35, 4, 1949, 0022-3476, 10.1016/s0022-3476(49)80063-1, 482–487, 18143940, but do require regeneration and may face challenges with absorption of undesired ions.

See also

• Aerobic denitrification
• Anaerobic respiration
• Bioremediation
• Climate change
• Hypoxia (environmental)
• Nitrogen fixation
• Simultaneous nitrification-denitrification

References

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