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
heterotrophicbacteria (such as Paracoccus denitrificans and various
pseudomonads),[1] although autotrophic denitrifiers have also been identified (e.g., Thiobacillus denitrificans).[2] Denitrifiers are represented in all main phylogenetic groups.[3] 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.[4] 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.[5]
Direct reduction from nitrate to
ammonium, a process known as dissimilatory nitrate reduction to ammonium or DNRA,[6] is also possible for organisms that have the nrf-
gene.[7][8] 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;[3] organisms identified as having these genes include Alcaligenes faecalis, Alcaligenes xylosoxidans, many in the genus Pseudomonas, Bradyrhizobium japonicum, and Blastobacter denitrificans.[9]
Overview
Half reactions
Denitrification generally proceeds through some combination of the following half reactions, with the enzyme catalyzing the reaction in parentheses:
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.[10] Typically, denitrification occurs in anoxic environments, where the concentration of dissolved and freely available oxygen is depleted. In these areas, nitrate (NO3−) or nitrite (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,[11]groundwater,[12]wetlands, oil reservoirs,[13] 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.[14] 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.[15] 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.[16]
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.[17] Moreover, the relative abundance of 14N can be analyzed to distinguish denitrification apart from other processes in nature.
Reduction under anoxic conditions can also occur through process called anaerobic ammonium oxidation (
anammox):[23]
NH4+ + NO2− → N2 + 2 H2O
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.[24] The microbial ecology of such engineered denitrification processes is determined by the nature of the electron donor and the process operating conditions.[25][26] Denitrification processes are also used in the treatment of
industrial wastewater.[27] 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.[16]
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.[28] 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,[29] and semi-conductors like TiO2.[30] 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,[31] but do require regeneration and may face challenges with absorption of undesired ions.
^Spanning, R., Delgado, M. and Richardson, D. (2005). "The Nitrogen Cycle: Denitrification and its Relationship to N2 Fixation". Nitrogen Fixation: Origins, Applications, and Research Progress. pp. 277–342.
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^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.
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^Scaglia, J.; Lensi, R.; Chalamet, A. (1985). "Relationship between photosynthesis and denitrification in planted soil". Plant and Soil. 84 (1): 37–43.
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^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". Environmental Microbiology. 18 (9): 2951–63.
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abJi, Bin; Yang, Kai; Zhu, Lei; Jiang, Yu; Wang, Hongyu; Zhou, Jun; Zhang, Huining (2015). "Aerobic denitrification: A review of important advances of the last 30 years". Biotechnology and Bioprocess Engineering. 20 (4): 643–651.
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^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.
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^Mulvaney, R. L.; Khan, S. A.; Mulvaney, C. S. (1997). "Nitrogen fertilizers promote denitrification". Biology and Fertility of Soils. 24 (2): 211–220.
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^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.
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^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.
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^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.
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^Rajmohan, K. S.; Gopinath, M.; Chetty, Raghuram (2016). "Review on challenges and opportunities in the removal of nitrate from wastewater using electrochemical method". 37. Triveni Enterprises: 1519–1528.
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