DENITRIFICATION

Denitrification is the reverse process of nitrogen fixation. In this process,

nitrates are reduced to atmospheric nitrogen by the action of
microorganisms in the soil.
Conditions/Requirements for the denitrification process
The conditions required for denitrification to take place are as follows:

Presence of facultative microorganisms

The microorganisms involved are either heterotrophic or autotrophic that can

switch to anaerobic condition when oxygen levels are depleted, and thus use
nitrate as electron acceptor to break down biodegradable substrates.
The microorganisms carrying out this role are referred to as denitrifiers and
some main examples are hiobacillus denitrificans, Micrococcus denitrificans,
and species of Pseudomonas, Bacillus, Achromobacter, etc. Generally,
denitrifiers are heterotrophic bacteria which use nitrate as electron acceptor
to break down biodegradable substrates, under anoxic conditions.

Presence of nitrate as electron acceptor and absence of dissolved

oxygen

It has been observed that a concentration of 0.2 to 0.5 mg/l of dissolved

oxygen reduces the rate of denitrification significantly. Thus, it is vital to
minimize the dissolved oxygen concentrations in order to maximize the
efficiency of the process. This can be achieved through a high level of
biochemical oxygen demand (BOD) and prevention of the aeration of the
wastewater. If oxygen is present, the latter will be used for metabolism
instead of nitrate. Therefore, in the absence of oxygen, nitrate becomes the
primary source of oxygen for the microorganisms.

pH and temperature

The optimum pH for denitrification lies between 7.0 and 8.5. As for
temperature, denitrification normally occurs between 35-50C.

Presence of a carbon source

A carbon source, that is, an organic compound is also required by the

denitrifiers to proceed with denitrification. The most commonly used carbon

source is methanol since it is readily decomposed under both anoxic and

aerobic conditions.
Steps involved in the denitrification process
Denitrification, also referred to as dissimilatory nitrate reduction, occurs in a
series of steps:
1. Nitrates are first reduced to nitrites, reaction catalyzed by dissimilatory
nitrate reductase.
2HNO3 ------> 2HNO2 +4e
2. Dissmilatory nitrite reductase then reduces nitrites to nitric oxide.
2HNO2 -----> 2NO +2e
3. Reduction of Nitric oxide to nitrous oxide by dissimilatory nitric oxide
reductase.
2NO ------>N2O +2e
4. Reduction of nitrous oxide to atmospheric nitrogen, a reaction
catalyzed by dissimilatory nitrous oxide reductase.
N2O ---------> N2 +2e
In this overall process, for every one mole of nitrate reduced, 24 molecules of
ATP are produced.
Organic denitrification
Organic denitrification is the process of denitrification involving an organic
material and it depends on the presence of facultative heterotrophs which
are able of using nitrate as their electron acceptor. While nitrates are
reduced to atmospheric nitrogen through several steps, the organic material
becomes oxidized to carbon dioxide and water. Organic substrates such as
methanol, ethanol and acetic acid can be used by the heterotrophic bacteria
under anoxic conditions.
Inorganic denitrification
Inorganic denitrification involves sulphide, ammonia and hydrogen as the
electron donor. However, in waste water, the concentrations of hydrogen and
sulphide are too low for them to be considered as electron donors. It has
been shown that ammonia could be an electron donor for the reduction of
nitrite to nitrogen gas. The ammonia undergoes oxidation reaction under

anoxic conditions and both ammonia and nitrite contribute equally to the
nitrogen content in the resultant nitrogen gas.
HNO2 + NH3 -----> N2 + 2H2O

References

http://agriinfo.in/?page=topic&superid=5&topicid=165 [Accessed on 11th

December 2012]
http://www.wastewaterhandbook.com/documents/nitrogen_removal/431_N
R_denitrification_prerequisites.pdf [Accessed on the 12th December 2012]
http://water.epa.gov/scitech/wastetech/upload/2008_01_23_mtb_etfs_deni
trifying.pdf [Accessed on 12th December 2012]
http://www.thewaterplanetcompany.com/docs/WPC_Nitrification%20&
%20Denitrification%20.pdf [Accessed on the 12th December 2012]
Bitton G., 2011. Wastewater Microbiology. John Wiley & Sons.
http://filebox.vt.edu/users/chagedor/biol_4684/Cycles/Denit.html
[Accessed on the 28th December 2012]