Denitrification

The processs 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 NO₃⁻ to N₂, and more than one enzymatic pathway has been identified in the reduction process. The denitrification process does not only provide energy to the organism performing nitrate reduction to dinitrogen gas, but also some anaerobic ciliates can use denitrifying endosymbionts to gain energy similar to the use of mitochondria in oxygen respiring organisms.

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.

Overview

Half reactions

Denitrification generally proceeds through some combination of the following half reactions, with the enzyme catalyzing the reaction in parentheses:

• NO₃⁻ + 2 H⁺ + 2 e⁻ → NO₂⁻ + H₂O (Nitrate reductase)
• NO₂⁻ + 2 H⁺ + e⁻ → NO + H₂O (Nitrite reductase)
• 2 NO + 2 H⁺ + 2 e⁻ → N₂O + H₂O (Nitric-oxide reductase)
• N₂O + 2 H⁺ + 2 e⁻ → N₂ + H₂O (Nitrous-oxide reductase)

The complete process can be expressed as a net balanced redox reaction, where nitrate (NO₃⁻) gets fully reduced to dinitrogen (N₂):

• 2 NO₃⁻ + 10 e⁻ + 12 H⁺ → N₂ + 6 H₂O

Conditions of denitrification

In nature, denitrification can take place in both terrestrial and marine ecosystems. Typically, denitrification occurs in anoxic environments, where the concentration of dissolved and freely available oxygen is depleted. In these areas, nitrate (NO₃⁻) or nitrite (NO₂⁻) can be used as a substitute terminal electron acceptor instead of oxygen (O₂), a more energetically favourable electron acceptor. Terminal electron acceptor is a compound that gets reduced in the reaction by receiving electrons. Examples of anoxic environments can include soils, groundwater, wetlands, oil reservoirs, poorly ventilated corners of the ocean and seafloor sediments.

Furthermore, denitrification can occur in oxic environments as well. High activity of denitrifiers can be observed in the intertidal zones, where the tidal cycles cause fluctuations of oxygen concentration in sandy coastal sediments. For example, the bacterial species Paracoccus denitrificans engages in denitrification under both oxic and anoxic conditions simultaneously. Upon oxygen exposure, the bacteria is able to utilize nitrous oxide reductase, an enzyme that catalyzes the last step of denitrification. Aerobic denitrifiers are mainly Gram-negative bacteria in the phylum Proteobacteria. Enzymes NapAB, NirS, NirK and NosZ are located in the periplasm, a wide space bordered by the cytoplasmic and the outer membrane in Gram-negative bacteria.

A variety of environmental factors can influence the rate of denitrification on an ecosystem-wide scale. For example, temperature and pH have been observed to impact denitrification rates. In the bacterial species, Pseudomonas mandelii, expression of denitrifying genes was reduced at temperatures below 30 °C and a pH below 5, while activity was largely unaffected between a pH of 6–8. Organic carbon as an electron donor is a common limiting nutrient for denitrification as observed in benthic sediments and wetlands. Nitrate and oxygen can also be potential limiting factors for denitrification, although the latter only has an observed limiting effect in wet soils. Oxygen likely affects denitrification in multiple ways—because most denitrifiers are facultative, oxygen can inhibit rates, but it can also stimulate denitrification by facilitating nitrification and the production of nitrate. In wetlands as well as deserts, moisture is an environmental limitation to rates of denitrification.

Additionally, environmental factors can also influence whether denitrification proceeds to completion, characterized by the complete reduction of NO₃⁻ to N₂ rather than releasing N₂O as an end product. Soil pH and texture are both factors that can moderate denitrification, with higher pH levels driving the reaction more to completion. Nutrient composition, particularly the ratio of carbon to nitrogen, is a strong contributor to complete denitrification, with a 2:1 ratio of C:N being able to facilitate full nitrate reduction regardless of temperature or carbon source. Copper, as a co-factor for nitrite reductase and nitrous-oxide reductase, also promoted complete denitrification when added as a supplement. Besides nutrients and terrain, microbial community composition can also affect the ratio of complete denitrification, with prokaryotic phyla Actinomycetota and Thermoproteota being responsible for greater release of N₂ than N₂O compared to other prokaryotes.

Denitrification can lead to a condition called isotopic fractionation in the soil environment. The two stable isotopes of nitrogen, ¹⁴N and ¹⁵N are both found in the sediment profiles. The lighter isotope of nitrogen, ¹⁴N, is preferred during denitrification, leaving the heavier nitrogen isotope, ¹⁵N, in the residual matter. This selectivity leads to the enrichment of ¹⁴N in the biomass compared to ¹⁵N. Moreover, the relative abundance of ¹⁴N can be analyzed to distinguish denitrification apart from other processes in nature.