# Denitrifying bacteria

Denitrifying bacteria are a diverse group of bacteria that encompass many different phyla. This group of bacteria, together with denitrifying fungi and archaea, is capable of performing denitrification as part of the nitrogen cycle.[1] They metabolise nitrogenous compounds using various enzymes, turning nitrogen oxides back to nitrogen gas or nitrous oxide.

## Diversity of denitrifying bacteria

There is a great diversity in bacteria capable of performing denitrification. Members of this group encompass most bacterial phyla and therefore possess a wide variety of physiological traits.[1] Denitrifying bacteria have been identified in over 50 genera with over 125 different species and are estimated to represent 10-15% of bacteria population in water, soil and sediment.[2] Denitrifying include for example several species of Pseudomonas, Alkaligenes , Bacillus and others. The majority of denitrifying bacteria are facultative aerobic heterotrophs that switch from aerobic respiration to denitrification when oxygen as an available terminal electron acceptor (TEA) runs out. This forces the organism to use nitrate to be used as a TEA.[1] Because the diversity of denitrifying bacteria is so large, this group can thrive in a wide range of habitats including some extreme environments such as environments that are highly saline and high in temperature.[1] Aerobic denitrifiers can conduct an aerobic respiratory process in which nitrate is converted gradually to N2 (NO3 →NO2 → NO → N2O → N2 ), using nitrate reductase (Nar or Nao), nitrite reductase (Nir), nitric oxide reductase (Nor), and nitrous oxide reductase (Nos). Phylogenetic analysis revealed that aerobic denitrifiers mainly belong to α-, β- and γ-Proteobacteria[3].

## Denitrification mechanism

Denitrifying bacteria use denitrification to generate ATP.[4]

The most common denitrification process is outlined below, with the nitrogen oxides being converted back to gaseous nitrogen:

2 NO3 + 10 e + 12 H+ → N2 + 6 H2O

The result is one molecule of nitrogen and six molecules of water. Denitrifying bacteria are a part of the N cycle, and consists of sending the N back into the atmosphere. The reaction above is the overall half reaction of the process of denitrification. The reaction can be further divided into different half reactions each requiring a specific enzyme. The transformation from nitrate to nitrite is performed by nitrate reductase (Nar)

NO3 + 2 H+ + 2 e → NO2 + H2O

Nitrite reductase (Nir) then converts nitrite into nitric oxide

2 NO2 + 4 H+ + 2 e → 2 NO + 2 H2O

Nitric oxide reductase (Nor) then converts nitric oxide into nitrous oxide

2 NO + 2 H+ + 2 e → N2O + H2O

Nitrous oxide reductase (Nos) terminates the reaction by converting nitrous oxide into dinitrogen

N2O + 2 H+ + 2 e → N2 + H2O

It is important to note that any of the products produced at any step can be exchanged with the soil environment.[4]

## Denitrifying bacteria and the environment

The process of denitrification can lower the fertility of soil as nitrogen, a growth-limiting factor, is removed from the soil and lost to the atmosphere. This loss of nitrogen to the atmosphere can eventually be regained via introduced nutrients, as part of the nitrogen cycle. Some nitrogen may also be fixated by species of nitrifying bacteria and the cyanobacteria. Another important environmental issue concerning denitrification is the fact that the process tends to produce large amounts of by-products. Examples of by-products are nitric oxide (NO) and nitrous oxide (N2O). NO is an ozone depleting species and N2O is a potent greenhouse gas which can contribute to global warming.[2]

## References

1. ^ a b c d Zumft, W. G. (1997). Cell biology and molecular basis of denitrification. Microbiology and Molecular Biology Reviews, 61(4), 533–616
2. ^ a b Eldor, A. (2015). Soil microbiology, ecology, and biochemistry (4th ed.). Chapter 14 Amsterdam: Elsevier.
3. ^ Ji, Bin; Yang, Kai; Zhu, Lei; Jiang, Yu; Wang, Hongyu; Zhou, Jun; Zhang, Huining (August 2015). "Aerobic denitrification: A review of important advances of the last 30 years". Biotechnology and Bioprocess Engineering. 20 (4): 643–651. doi:10.1007/s12257-015-0009-0. ISSN 1226-8372.
4. ^ a b Bothe, H., Ferguson, S., & Newton, W. (2007). Biology of the nitrogen cycle. Amsterdam: Elsevier.
Aerobic denitrification

Aerobic denitrification or co-respiration the simultaneous use of both oxygen (O2) and nitrate (NO3−) as oxidizing agents, performed by various genera of microorganisms. This process differs from anaerobic denitrification not only in its insensitivity to the presence of oxygen, but also in that it has a higher potential to create the harmful byproduct nitrous oxide.Nitrogen, acting as an oxidant, is therefore reduced in a succession of four reactions performed by the enzymes nitrate, nitrite, nitric-oxide, and nitrous oxide reductases. The pathway ultimately yields reduced molecular nitrogen (N2), as well as, when the reaction does not reach completion, the intermediate species nitrous oxide (N2O). A simple denitrification reaction proceeds as:

NO3− → NO2− → NO → N2O → N2 (g)The respiration reaction which utilizes oxygen as the oxidant is:

C6H12O6 (aq) + 6O2 (g) → 6CO2 (g) + 6H2OClassically, it was thought that denitrification would not occur in the presence of oxygen since there seems to be no energetic advantage to using nitrate as an oxidant when oxygen is available. Experiments have since proven that denitrifiers are often facultative anaerobes and that aerobic denitrification does indeed occur in a broad range of microbial organisms with varying levels of productivity, usually lower productivity than results from purely aerobic respiration. The advantages of being able to perform denitrification in the presence of oxygen are uncertain, though it is possible that the ability to adapt to changes in oxygen levels plays a role. Aerobic denitrification may be found in environments where fluctuating oxygen concentrations and reduced carbon are available. The relative harsh environment inspires the potential of denitrifiers to degrade toxic nitrate or nitrate under an aerobic atmosphere. Aerobic denitrifiers tend to work efficiently at 25 ~ 37°C and pH 7 ~ 8, when dissolved oxygen concentration is 3 ~ 5 mg/L and C/N load ratio is 5 ~ 10.

Aromatoleum

"Aromatoleum" is a genus of proteobacteria capable of microbial biodegradation of organic pollutants. It has one single described species member, A. aromaticum, for which the only strain is strain EbN1.

This taxonomy is accepted by the NCBI taxonomy database, and consequently by many bioinfomatic databases. However, it the strain EbN1 has not been described in detail, therefore, according to the International Code of Nomenclature of Bacteria, the name "Aromatoleum aromaticum" is not valid and should be officially referred to as Azoarcus sp. EbN1 as it belongs to the Azoarcus/Thauera cluster. The discovery of the strain was published in 1995, and was subsequently referred to in the literature as "Aromatoleum aromaticum" and cited as "(Rabus, unpublished data)".A. aromaticum strain EbN1 has been fully sequenced by the same researchers who discovered it and coworkers. It has one chromosome and two plasmids, encoding for 10 anaerobic and 4 aerobic aromatic degradation pathways. The genome is rich in paralogous gene clusterss, mobile gene elements, and genes similar to that from other bacteria, suggesting a history full of horizontal gene transfer events. The bacterium has a well-regulated metabolic network. Unlike many species in Azoarcus proper, it is incapable of fixing nitrogen.

Azoarcus toluclasticus

Azoarcus toluclasticus is a gram negative bacterium from the genus of Azoarcus.

Azoarcus toluvorans

Azoarcus toluvorans is a bacterium from the genus of Azoarcus.

Bacillus azotoformans

Bacillus azotoformans is a species of bacteria within the genus Bacillus. Novel nitrite reductases have been isolated from strains of this species.

Benzoate—CoA ligase

In enzymology, a benzoate-CoA ligase (EC 6.2.1.25) is an enzyme that catalyzes the chemical reaction

ATP + benzoate + CoA ${\displaystyle \rightleftharpoons }$ AMP + diphosphate + benzoyl-CoA

The 3 substrates of this enzyme are ATP, benzoate, and CoA, whereas its 3 products are AMP, diphosphate, and benzoyl-CoA.

This enzyme belongs to the family of ligases, specifically those forming carbon-sulfur bonds as acid-thiol ligases. The systematic name of this enzyme class is benzoate:CoA ligase (AMP-forming). Other names in common use include benzoate-coenzyme A ligase, benzoyl-coenzyme A synthetase, and benzoyl CoA synthetase (AMP forming). This enzyme participates in benzoate degradation via coa ligation.

"Candidatus Brocadia anammoxidans" is a bacterial member of the order Planctomycetes and therefore lacks peptidoglycan in its cell wall, and has a compartmentalized cytoplasm.

"Candidatus Brocadia anammoxidans" was the first discovered organism capable of the anaerobic oxidation of ammonium, and it is the only organism known to produce hydrazine. This process (dubbed the anammox-process) was discovered in the 1980s by the Gijs Kuenen lab in a waste water treatment plant in Delft, Netherlands. Ammonium oxidation is coupled to nitrite reduction to form the harmless dinitrogen gas.

The key enzyme involved in this reaction, hydroxylamine oxidoreductase, is located in an organelle-like structure called the anammoxosome. The ability to oxidize ammonium anaerobically makes "Candidatus Brocadia anammoxidans" potentially useful for reducing—or eliminating—ammonium from waste water.

Cattle urine patches

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.

Denitrification

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.

Ethylbenzene

Ethylbenzene is an organic compound with the formula C6H5CH2CH3. It is a highly flammable, colorless liquid with an odor similar to that of gasoline. This monocyclic aromatic hydrocarbon is important in the petrochemical industry as an intermediate in the production of styrene, the precursor to polystyrene, a common plastic material. In 2012, more than 99% of ethylbenzene produced was consumed in the production of styrene.

Gijs Kuenen

Johannes Gijsbrecht Kuenen (born 9 December 1940, Heemstede) is a Dutch microbiologist who is professor emeritus at the Delft University of Technology and a visiting scientist at the University of Southern California. His research is influenced by, and a contribution to, the scientific tradition of the Delft School of Microbiology.

Kuenen studied at the University of Groningen, where he received both his Doctorandus degree and in 1972 his Doctorate (PhD) under the supervision of Professor Dr. Hans Veldkamp. The title of his thesis was ‘‘Colourless sulphur bacteria from Dutch tidal mudflats’’. After a short post-doc at the University of California in Los Angeles (USA), he returned as a senior lecturer to Groningen. In 1980, he moved to Delft to become the 4th Professor of Microbiology (succeeding M.W. Beijerinck and A.J. Kluyver) at Delft University of Technology. Kuenen's initial research interests were (the application of) bacteria involved in the natural sulfur cycle and yeast physiology and metabolism. His later interest in the (eco)physiology of nitrifying and denitrifying bacteria has led a.o. to the discovery of the bacteria within the phylum planctomycetes that perform the Anammox process. In addition, his research has been focussed on (halo) alkaliphilic sulfur-oxidizing bacteria from soda lakes. Gijs Kuenen retired in 2005 but remains active in science.

Halospina

Halospina is an extremely halophilic genus of bacteria from the family of Hahellaceae with one known species (Halospina denitrificans). Halospina denitrificans has been isolated from sediments from a hypersaline lake.

Metabolic waste

Metabolic wastes or excretements are substances left over from metabolic processes (such as cellular respiration) which cannot be used by the organism (they are surplus or toxic), and must therefore be excreted. This includes nitrogen compounds, water, CO2, phosphates, sulphates, etc. Animals treat these compounds as excretes. Plants have chemical "machinery" which transforms some of them (primarily the nitrogen compounds) into useful substances, and it has been shown by Brian J. Ford that abscised leaves also carry wastes away from the parent plant. In this way, Ford argues that the shed leaf acts as an excretory (an organ carrying away excretory products).

All the metabolic wastes are excreted in a form of water solutes through the excretory organs (nephridia, Malpighian tubules, kidneys), with the exception of CO2, which is excreted together with the water vapor throughout the lungs. The elimination of these compounds enables the chemical homeostasis of the organism.

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).

Pimeloyl-CoA dehydrogenase

In enzymology, a pimeloyl-CoA dehydrogenase (EC 1.3.1.62) is an enzyme that catalyzes the chemical reaction

pimeloyl-CoA + NAD+ ${\displaystyle \rightleftharpoons }$ 6-carboxyhex-2-enoyl-CoA + NADH + H+

Thus, the two substrates of this enzyme are pimeloyl-CoA and NAD+, whereas its 3 products are 6-carboxyhex-2-enoyl-CoA, NADH, and H+.

This enzyme belongs to the family of oxidoreductases, specifically those acting on the CH-CH group of donor with NAD+ or NADP+ as acceptor. The systematic name of this enzyme class is pimeloyl-CoA:NAD+ oxidoreductase. This enzyme participates in benzoate degradation via coa ligation.

Refugium (fishkeeping)

In fishkeeping, a refugium is an appendage to a marine, brackish, or freshwater fish tank that shares the same water supply. It is a separate sump, connected to the main show tank. It is a "refugium" in the sense that it permits organisms to be maintained that would not survive in the main system, whether food animals, anaerobic denitrifying bacteria, or photosynthesizers. For some applications water flow is limited in order to protect plants or animals that require slow flow. The refugium light cycle can be operated opposite to the main tank, in order to keep total system pH more stable (due to the uptake of acid-forming CO2 by photosynthesis occurring in the refugium during its "daylight" hours). One volume guideline for a refugium is 1:10 main tank volume.

A refugium may be used for one or more purposes such as denitrification, nutrient export, plankton production, circulation, surface agitation to improve oxygen exchange with the atmosphere or even aesthetic purposes.

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, concentration

Water stagnation

Water stagnation occurs when water stops flowing. Stagnant water can be a major environmental hazard.

Zobellella denitrificans

Zobellella denitrificans is a Gram-negative, facultatively anaerobic, heterotrophic and denitrifying bacterium from the genus of Zobellella which has been isolated from sediments from a mangrove ecosystems from Miaoli County in Taiwan.

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