Denitrification: Nitrogen is one of the crucial nutrients required to produce important components like amino acids, proteins, nucleic acids, etc. Even huge trees, all the other plants need a sufficient annual supply for proper growth and production. Nitrogen is primarily absorbed through fine roots as either ammonium or nitrate. In this article, we will learn about denitrification, its reactions, the complete process of Denitrification and the factors that affect this process.

What is Denitrification Process?

The phenomenon of reduction of nitrates and nitrites into atmospheric nitrogen or as an oxide of nitrogen by the action of denitrifying bacteria is known as Denitrification. In this process, nitrogen in the combined state is reduced to its gaseous state. Combined nitrogen is the nitrogen along with any other element, e.g., nitrate, nitrite, ammonium, amines, etc. This process is facilitated by microorganisms. The common organisms that facilitate denitrification are facultative anaerobic bacteria. Denitrification is a process of respiration and the acquisition of energy for these bacteria.

Fig:  Bacteria involved in Nitrogen Cycle

Reaction Involved in Denitrification

Denitrification is an anaerobic process, it utilizes nitrate as an electron acceptor. Denitrification is usually a stepwise reduction of the following intermediates:
\({\rm{N}}{{\rm{O}}_3}^ –  \to {\rm{N}}{{\rm{O}}_2}^ –  \to {\rm{NO}} \to {{\rm{N}}_2}{\rm{O}} \to {{\rm{N}}_2}\)

In general, several species of anaerobic bacteria are involved in the complete reduction of nitrate into molecular or atmospheric nitrogen.
There are many enzymatic pathways involved in this reduction process. Complete denitrification is a very significant process for the environment as the intermediates produced during denitrification are very important ecologically and some intermediates like nitric oxide and nitrous oxide are significant greenhouse gases. These gases interact with the ozone and UV radiations from the sun and produce nitric acid, a component of acid rain.

Denitrification is a microbially facilitated process because facultative anaerobes use nitrate and nitrite like oxygen for their respiration and other enzymatic reactions since it has a high reduction potential. It involves stepwise reduction of nitrate \(\left( {{\rm{N}}{{\rm{O}}_{\rm{3}}}^{\rm{ – }}} \right)\) to nitrite \(\left( {{\rm{N}}{{\rm{O}}_{\rm{2}}}^{\rm{ – }}} \right)\) to nitric oxide \(\left( {{\rm{NO}}} \right)\) to nitrous oxide \(\left( {{{\rm{N}}_{\rm{2}}}{\rm{O}}} \right){\rm{,}}\) and finally to atmospheric dinitrogen \(\left( {{{\rm{N}}_{\rm{2}}}} \right)\) by the action of the following enzymes:

Nitrate reductase, nitrite reductase, nitric oxide reductase, and nitrous oxide reductase.

The overall denitrification process is a redox reaction which can be written as:

\({\rm{2N}}{{\rm{O}}_3}^ –  + {\rm{10}}{{\rm{e}}^ – } + 12{{\rm{H}}^ + } \to {{\rm{N}}_2} + 6{{\rm{H}}_2}{\rm{O}}\)

Denitrification Process in the Nitrogen Cycle

The steps of the nitrogen cycle are nitrogen fixation, nitrification, nitrogen assimilation, ammonification, and denitrification. Let us study in brief the nitrogen cycle before studying denitrification in detail.
1. Nitrogen Fixation: It is the process of fixing atmospheric nitrogen into the soil mainly through biological processes via bacteria or sometimes by physical processes which require a lot of energy with the release of enormous amounts of heat, pressure, and energy. In this process, nitrogen is converted to either ammonia or nitrate.
2. Nitrification: It is a two step process where the first reaction is the conversion of ammonia into nitrate and the second reaction is the further conversion of nitrate into nitrite.
3. Nitrogen Assimilation: The process in which the nitrates and ammonia produced in the process of nitrogen fixation and nitrification are assimilated or incorporated or absorbed by plants and animals is referred to as nitrogen assimilation.
4. Ammonification: Large amounts of organic nitrogen compounds are formed during assimilation like amino acids, proteins, and nucleic acids. In this process, these organic nitrogen are converted back to ammonia which is then available for either assimilation or nitrification.
5. Denitrification: Denitrification is the process of the reduction of nitrates or nitrites into gaseous nitrogen by facultative anaerobes. This process usually takes place when there is no oxygen or very little oxygen in the environment such as in the deep soil near the water table. Therefore, wetlands are the areas where excess nitrogen is reduced through denitrification processes.
Since, nitrogen is a source of energy for most anaerobes, this is a significant process in the nitrogen cycle. In wet soils, these soil bacteria convert nitrates and nitrites into various nitrogen gases like nitrous oxide, nitric oxide, etc., as a result of their reduction mechanisms by the action of several enzymes.

Fig: Stages involved in Nitrogen Cycle

Dissimilatory Nitrate Reduction to Ammonium (or DNRA) is a process of direct reduction from nitrate to ammonia, which is carried out by organisms having the nif– gene. This is accomplished by certain chemo organoheterotroph bacteria like Beggiatoa and the benthic diatom Amphora coffeaeformis. This process converts the nitrate to ammonia and not to molecular nitrogen. This increases the sustainable primary production and nitrification.

Differences between Nitrification, Ammonification and Denitrification

Nitrification	Ammonification	Denitrification
Conversion of ammonium \({\rm{N}}{{\rm{H}}_{\rm{4}}}^{\rm{ + }}\) into nitrate \(\left( {{\rm{N}}{{\rm{O}}_{\rm{3}}}^{\rm{ – }}} \right){\rm{.}}\)	Conversion of nitrate \(\left( {{\rm{N}}{{\rm{O}}_{\rm{3}}}^{\rm{ – }}} \right)\) and nitrite \(\left( {{\rm{N}}{{\rm{O}}_2}^{\rm{ – }}} \right)\) to ammonia \(\left( {{\rm{N}}{{\rm{H}}_{\rm{3}}}} \right){\rm{.}}\)	Conversion of nitrate \(\left( {{\rm{N}}{{\rm{O}}_{\rm{2}}}^{\rm{ – }}} \right)\) and nitrite \(\left( {{\rm{N}}{{\rm{O}}_{\rm{3}}}^{\rm{ – }}} \right)\) to gaseous nitrogen \(\left( {{{\rm{N}}_{\rm{2}}}} \right){\rm{.}}\)
Carried by Nitrifying bacteria like Nitrobacter, Nitrosomonas, Nitrosococcus, Nitrococcus, Nitrospira, etc.	Performed by Ammonifying bacteria like Bacillus, Clostridium, Proteus, Pseudomonas, and Streptomyces.	Denitrifying bacteria like Lactobacillus, Pseudomonas, Thiobacillus, Spirillum, Thiobacillus, etc.
These are autotrophic bacteria and require aerobic conditions.	These are heterotrophic bacteria and require both aerobic and anaerobic conditions.	These are also heterotrophic bacteria and require anaerobic conditions.
The precursor for the process is ammonium \(\left( {{\rm{N}}{{\rm{H}}_{\rm{4}}}^{\rm{ + }}} \right){\rm{.}}\) The end product is nitrate \(\left( {{\rm{NO}}_{\rm{3}}^{\rm{ – }}} \right){\rm{.}}\)	The precursors for this process are nitrate \(\left( {{\rm{N}}{{\rm{O}}_{\rm{3}}}^{\rm{ – }}} \right)\) and nitrite \(\left( {{\rm{N}}{{\rm{O}}_{\rm{2}}}^{\rm{ – }}} \right){\rm{.}}\) The end product is ammonia.	The precursor for the process is nitrate \(\left( {{\rm{N}}{{\rm{O}}_{\rm{3}}}^{\rm{ – }}} \right){\rm{.}}\) The end product is nitrogen gas.
This process provides nitrate to the plants which act as a significant nitrogen source.	It helps to clean up the environment by degrading plant and animal matter.	It is used in industrial or sewage wastewater treatment plants and is beneficial for aquatic environments.

Microorganisms Involved in Denitrification

The bacteria involved in denitrification are known as denitrifying bacteria or denitrifiers. Denitrifying bacteria include \(10-15%\) of the bacterial population. These are present in the soil, water, and sediments.

Few examples include Thiobacillus denitrificans, Micrococcus denitrificans, species of Pseudomonas, Clostridium, Bacillus, Serratia, and Achromobacter. Pseudomonas aeruginosa acts as a denitrifier in waterlogged areas. Certain fungi such as Fusarium, Trichoderma, and 67 strains of yeast, among the most widely found, and, Candida, Saccharomycopsis and Trichosporon are also good denitrifiers.

Factors Affecting Denitrification

Various factors govern the process of denitrification such as pH, temperature, hydraulic conditions, quantity and quality of organic carbon content, microbial population, and nitrate concentration.
1. pH– The rate of denitrification is greatly affected by soil pH. The rate of denitrification is very low below the pH of \(4.8\) and the rate increases with an increase in soil pH and the reactions were very rapid at a \({\rm{pH}}\) range of \(8.0\) to \(8.6.\)
2. Temperature– The rate of denitrification is raised rapidly with an increase in temperature from \({{\rm{2}}^{\rm{o}}}{\rm{C}}\) to \({\rm{2}}{{\rm{5}}^{\rm{o}}}{\rm{C}}{\rm{.}}\) The optimum temperature for the process of denitrification is about \({\rm{6}}{{\rm{0}}^{\rm{o}}}{\rm{C}}{\rm{.}}\)
3. Hydraulic conditions– The degree of the water content (moisture content) of the soil has a deep influence on the rate of denitrification. Lower the moisture content, lower the process of denitrification. The higher the moisture content, the greater is the rate of denitrification, up to a certain level. The critical moisture content in the soil must be \(60%\) of the water holding capacity of the soil.
4. Quantity and quality of organic content– Carbon content plays a very important role in the denitrification process. The quality and quantity of carbon content greatly affect the growth of denitrifying bacteria which in contrast affects denitrification. The greater the organic content in the soil the more is the growth of denitrifiers, and therefore, denitrification progresses rapidly and vice versa. A similar thesis applies to nitrate concentration as well.
5. Microbial communities– The number of microbes greatly affects the process of denitrification. Under optimum conditions, microbes grow luxuriantly. As the number of bacteria increases, the rate of denitrification increases tremendously as well.

Significance of Denitrification

Following are some of the significance of denitrification in our environment:
1. Denitrification maintains homeostasis in the environment, although it is naturally unfavorable for soil fertility and plant growth it maintains the balance in the soil by converting nitrates into gaseous nitrogen thereby maintaining the concentration of atmospheric nitrogen content.
2. It plays a vital role in industrial and sewage water treatment by scavenging all the waste materials containing nitrogen content.
3. Also helps in maintaining nitrogen concentration in aquatic environments as well.
4. Since, this process converts nitrates into atmospheric nitrogen, it depletes fertility in the soil, thereby, reducing agricultural productivity.

Summary

Nitrogen is a key nutrient for both plants and animals and if it is not managed carefully, may lead to serious environmental problems. It is replenished through the nitrogen cycle. Denitrification is a crucial step of the nitrogen cycle.  Denitrification occurs under special conditions in both terrestrial and marine ecosystems. In simple words, it takes place where oxygen is depleted and bacteria respire nitrate as a substitute terminal electron acceptor.

Oxygen in our atmosphere is present in high concentrations, denitrification only occurs in anaerobic environments where oxygen consumption surpasses the oxygen supply and where ample quantities of nitrate are present. These environments may comprise certain soils and groundwater, wetlands, oil reservoirs, poorly ventilated corners of the ocean, and in seafloor sediments.

Frequently Asked Questions (FAQs) on Denitrification

Q.1. What happens during denitrification?
Ans: Denitrification is the process of the reduction of nitrates or nitrites into gaseous nitrogen with the help of facultative anaerobic bacterias.

Q.2. What is the importance of denitrification?
Ans: Denitrification is an essential step of the nitrogen cycle which is one of the most nutrient cycles in the environment to maintain the balance of nitrogen in the soil by converting nitrates into gaseous nitrogen. It plays a vital role in industrial and sewage water treatment by scavenging all the waste materials containing nitrogen content.

Q.3. What is the denitrification equation?
Ans: The denitrification process is a redox reaction which can be written as:
\({\rm{2N}}{{\rm{O}}_3}^ –  + {\rm{10}}{{\rm{e}}^ – } + 12{{\rm{H}}^ + } \to {{\rm{N}}_2} + 6{{\rm{H}}_2}{\rm{O}}\)

Q.4. What is the difference between nitrification and denitrification?
Ans: Nitrification and denitrification are the two important processes of the nitrogen cycle. In the process of Nitrification, nitrifying bacteria oxidise ammonia to nitrite which is further oxidised into nitrate. On the other hand, denitrification is the opposite of nitrification. In the denitrification process, microorganisms reduce nitrate back to nitrogen.

Q.5. What is the difference between Denitrification and Ammonification?
Ans: Denitrification process is carried by bacteria, they convert nitrates \(\left( {{\rm{N}}{{\rm{O}}_{\rm{3}}}^{\rm{ – }}} \right)\) to nitrogen gas \(\left( {{{\rm{N}}_{\rm{2}}}} \right){\rm{.}}\) Whereas, ammonification is the process of converting organic nitrogenous matter from living organisms into ammonium \(\left( {{\rm{N}}{{\rm{H}}_{\rm{4}}}^{\rm{ + }}} \right){\rm{.}}\)

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