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), although autotrophic denitrifiers have also been identified (e.g., Thiobacillus denitrificans). Denitrifiers are represented in all main phylogenetic groups. Generally several species of bacteria are involved in the complete reduction of nitrate to N2, and more than one enzymatic pathway has been identified in the reduction process.
Direct reduction from nitrate to ammonium, a process known as dissimilatory nitrate reduction to ammonium or DNRA, is also possible for organisms that have the nrf-gene. This is less common than denitrification in most ecosystems as a means of nitrate reduction. Other genes known in microorganisms which denitrify include nir (nitrite reductase) and nos (nitrous oxide reductase) among others; organisms identified as having these genes include Alcaligenes faecalis, Alcaligenes xylosoxidans, many in the genus Pseudomonas, Bradyrhizobium japonicum, and Blastobacter denitrificans.
Denitrification generally proceeds through some combination of the following half reactions, with the enzyme catalyzing the reaction in parentheses:
- NO3− + 2 H+ + 2 e−→ NO
2− + H2O (Nitrate reductase)
2− + 2 H+ + e− → NO + H2O (Nitrite reductase)
- 2 NO + 2 H+ + 2 e− → N
2O + H2O (Nitric oxide reductase)
2O + 2 H+ + 2 e− → N
2 + H2O (Nitrous oxide reductase)
- 2 NO3− + 10 e− + 12 H+ → N2 + 6 H2O
Conditions of denitrification
In nature, denitrification can take place in both terrestrial and marine ecosystems. 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, groundwater, wetlands, oil reservoirs, 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. 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. 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.
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. Moreover, the relative abundance of 14N can be analyzed to distinguish denitrification apart from other processes in nature.
Use in wastewater treatment
Denitrification is commonly used to remove nitrogen from sewage and municipal wastewater. It is also an instrumental process in constructed wetlands and riparian zones for the prevention of groundwater pollution with nitrate resulting from excessive agricultural or residential fertilizer usage. Wood chip bioreactors have been studied since the 2000s and are effective in removing nitrate from agricultural run off and even manure.
- 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. The microbial ecology of such engineered denitrification processes is determined by the nature of the electron donor and the process operating conditions. Denitrification processes are also used in the treatment of industrial wastewater. 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.
- Aerobic denitrification
- Anaerobic respiration
- Climate change
- Hypoxia (environmental)
- Nitrogen fixation
- Simultaneous nitrification-denitrification
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