Wastewater Treatment Fundamentals: What Is Denitrification?

The overall denitrification process can be represented by the following equation:

2 NO3 + 10 e + 12 H → N2 + 6 H2O, ΔG = −333 kJ/mol

It comprises the following four reduction reactions:

Nitrate (NO3) is reduced to nitrite (NO2): 2 NO3 + 4 H + 4 e → 2 NO2 + 2 H2O

Nitrite (NO2) is reduced to nitric oxide (NO): 2 NO2 + 4 H + 2 e → 2 NO + 2 H2O

Nitric oxide (NO) is reduced to nitrous oxide (N2O): 2 NO + 2 H + 2 e → N2O + H2O

Nitrous oxide (N2O) is reduced to nitrogen gas (N2): N2O + 2 H + 2 e → N2 + H2O

All four reactions above are exothermic. Therefore, under anoxic or oxygen-free conditions, bacteria can use nitrate (NO3) as the terminal electron acceptor (TEA) of the electron transport chain (ETC) to complete material and energy exchange.

Significance and Impact for Nature and Humanity

Denitrification is highly significant in nature and is a key link in the nitrogen cycle. It reduces the NO3- that leaches from soil into rivers and oceans, eliminating the toxic effects of nitrate accumulation on living organisms. Together with anaerobic ammonium oxidation (Anammox), it forms the pathway by which nitrogen fixed in nature returns to the atmosphere.

In agriculture, denitrification reduces nitrate to nitrogen gas, thereby lowering the nitrogen nutrient content of the soil, which is unfavourable for crop production. Farmers therefore often carry out inter-row loosening of the soil to prevent denitrification.

In environmental protection, denitrification together with nitrification forms various process configurations and is the principal method of biological nitrogen removal, widely applied in wastewater treatment plants around the world. The denitrifying bacteria used in wastewater treatment are heterotrophs; they grow rapidly but require an external organic carbon source. In practice, a small amount of methanol or other organic matter is sometimes dosed to ensure the denitrification process proceeds smoothly.

Denitrifying bacteria are widely distributed in nature in various forms, such as:

Paracoccus denitrificans (autotrophic, oxidises hydrogen gas, H2)

Thiobacillus denitrificans (autotrophic, oxidises sulfide (S) or thiosulfate (S2O3))

Pseudomonas stutzeri (heterotrophic, oxidises organic carbon)

Denitrifying bacteria are mainly prokaryotes and are abundant in the α-, β- and γ-Proteobacteria. Known denitrifying genera include Achromobacter, Acinetobacter, Agrobacterium, Bacillus, Chromobacterium, Flavobacterium, Spirillum, Vibrio, Halobacterium, Methanomonas, Pseudomonas and others.

A few denitrifiers are autotrophs, such as Thiobacillus denitrificans; they obtain energy by oxidising sulfur or nitrate, assimilate carbon dioxide, and use nitrate as the terminal electron acceptor of respiration.

Another use is to employ NO2- and NO3- as the terminal electron acceptors of respiration, reducing nitrate to nitrogen (N2); this is called denitrification or nitrogen removal: NO3- → NO2- → N2↑. Only a minority of bacteria can carry out denitrification, and this physiological group is known as the denitrifiers. Most denitrifying bacteria are heterotrophs, such as Micrococcus denitrificans and Pseudomonas denitrificans, which use organic matter as their nitrogen source and energy source and perform anaerobic respiration.

Microorganisms take up and utilise nitrate for two entirely different purposes. One is to use the nitrogen it contains as a nitrogen source, known as assimilatory nitrate reduction: NO3- → NH4+ → organic nitrogen. Many bacteria, actinomycetes and moulds can use nitrate as a nitrogen nutrient.