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Denitrification is a microbially facilitated process of nitrate reduction (performed

by a large group of heterotrophic facultative anaerobic bacteria) that may
ultimately produce molecular nitrogen (N2) through a series of intermediate
gaseous nitrogen oxide products (NO3- NO2- NO + N2O N2 (g)). This
respiratory process reduces oxidized forms of nitrogen in response to the oxidation
of an electron donor such as organic matter (methanol, ethanol, glucose, acetate
The preferred nitrogen electron accepters 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.
Denitrification proceeds in a stepwise manner in which nitrate (NO3-) is
sequentially reduced to nitrite (NO2-), nitric oxide (NO), and nitrous oxide
(N2O) and N2 gas.

Where it takes place and why?

Denitrification takes place under special conditions in both terrestrial and marine
ecosystems. In general. It occurs where oxygen, (a more energetically favourable
electron acceptor) is depleted and so bacteria respire nitrate as a substitute
terminal electron acceptor.
Due to high concentration of oxygen in our atmosphere denitrification only takes
place in anoxic (reduced supply of oxygen) environments where oxygen
consumptions exceeds the oxygen supply and where sufficient quantity of
nitrate is present. These environments may include soil, groundwater,
wetlands, oil reservoirs, poorly ventilated corners of the ocean and in
seafloor sediments
Denitrifiers (denitrifying bacteria)
The microbes which denitrifies the oxidized forms of nitrogen into
molecular nitrogen is called denitrifers. Denitrifiers are chemotrophs; utilizing
organic and inorganic electron donors. Those that utilize organic electron donors
are heterotrophs and are widespread among Proteobacteria.
Denitrification is performed primarily by
heterotrophic bacteria such as Paracoccus denitrifers, Pseudomonads,
autotrophic denitrifiers such as Thiobacillus denitrificans
halophilic Archaea such as Halobacterium
They are commonly found in soil, sediments, surface water, groundwater and
wastewater treatment plants etc.

Theses microorganisms contain specific genes namely nfr genes (nitrate

reductase), nir (nitrite reductase) genes and nos (nitrous oxide
reductase) genes for denitrification.
All the denitrifiers are facultative aerobes, which means that they shift (covert)
NO3- or NO2- respiration when O2 becomes limiting. Denitrifying microbes
require a very low oxygen concentration of less than 10%, as well as organic
C for energy.

Factors influencing denitrification

1. It occurs where oxygen consumptions exceeds the oxygen supply (oxygen
depletion) and sufficient quantity of nitrate is present
2. Oxygen concentration controls denitrification in two ways;
a. High concentration of dissolved oxygen represses nitrogen-reductase genes in
the bacteria e.g. nitrogen reductase genes in Pseudomonas stutzeri are
repressed by D.O conc greater than 2.5 to 5 mg O2/ L
b. Very high concentration (tenths of mg O2/L) D.O inhibits activity of
3. Very Low concentration of the election donor (i.e. high concentration of D.O)
leads to accumulation of denitrified intermediate gaseous nitrogen oxide products
(NO2-, NO, N2O), whereas low concentration of election donor limits the supply of
electrons to drive the reductive half-reactions.
4. pH above 7 to 8 leads to accumulation of intermediates

1. In environmental biotechnology denitrification is applied when the complete
removal of N is required. Which includes
Advanced treatment of wastewater discharged to watersheds that
must be protected from eutrophication.
Treatment of high N content waste such as agricultural runoff and
wastewater from feedlots.
Treatment of drinking water that contains elevated NO3- and NO2 To remove nitrogen from sewage and municipal waste
To prevent groundwater pollution from nitrate excessive agricultural
fertilizer usage.
Used to treat industrial wastewater
Since denitrification can lower leaching of NO3 to groundwater, it can be
strategically used to treat sewage or animal residues of high nitrogen content.


Denitrification allows for the production of N20, which is a greenhouse gas that has
considerable influence on global warming.

Types of denitrification
Denitrification process can be divided into two major classes; tertiary
denitrification and one sludge denitrification based on exogenous donor
Tertiary denitrification requires the addition of exogenous donor whereas one
sludge denitrification uses already present electron donor in the wastewater.
Tertiary denitrification

Tertiary denitrification is carried out in the places like agricultural areas and
drinking water treatment plants and wastewater treatment plants
where water to be treated contains nitrate and nitrate and little or no electron
donors. This situation occurs naturally by agricultural runoff with high level
of nitrogen content fertilizers. Drinking water supplies in agricultural
regions also contain high NO3- level but little electron donor.
Tertiary denitrification follows the aerobic biological process (i.e. secondary
treatment of wastewater (sewage) treatment). In the secondary treatment