Nitrification

The biological oxidation of ammonia to nitrite, followed by the oxidation of nitrite to nitrate by different organisms or direct ammonia oxidation to nitrate in comammox bacteria, is known as nitrification. The rate-limiting stage of the nitrification reaction process is generally the conversion of ammonia to nitrite. In the nitrogen cycle in soil, nitrification is a crucial phase. Small groups of autotrophic nitrification bacteria and archaea undertake nitrification, which is an aerobic process. The image of the nitrogen cycle is given below which also shows the process of nitrification.

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The Discovery of Nitrification

Louis Pasteur was the first to propose that the oxidation of ammonia to nitrate is a biological process in 1862. In 1875, during a quality assessment of water from Berlin wells, Alexander Müller discovered that ammonium was stable in sterilized solutions but nitrified in natural waters. According to A. Müller, microorganisms are responsible for nitrification. In 1877, two French agricultural chemists working in Paris, Jean-Jacques Schloesing and Achille Müntz, showed that nitrification is actually a microbially mediated process through studies with liquid sewage and artificial soil matrix (sterilized sand with powdered chalk).

Robert Warrington, who was examining the nitrification capabilities of garden soil at the Rothamsted experimental station in Harpenden, England, verified their findings in 1878. In 1879, R. Warrington made the initial discovery that nitrification is a two-step process, which John Munro verified in 1886. Although it was often thought that two-step nitrification was divided into multiple life phases or character qualities of a single microbe, this was not the case.

Percy and Grace Frankland, two English scientists from Scotland, most likely discovered the first pure nitrifier (ammonia-oxidizing) in 1890. Before that, only enrichment nitrifying cultures, not pure ones, could be established by Warrington, Sergei Winogradsky, and the Frankland's. With a strategy of successive dilutions, low inoculum, and extended culture durations measured in years, Frankland and Frankland were successful. In the same year (1890), Sergei Winogradsky claimed pure culture separation, although his culture was still a co-culture of ammonia- and nitrite-oxidizing bacteria. S. Winogradsky was successful a year later, in 1891. 

In reality, during the serial dilutions, ammonia-oxidizers and nitrite-oxidizers were inadvertently separated, resulting in a pure culture capable of solely ammonia oxidation. As a result, according to Frankland and Frankland, these pure cultures lose their capacity to accomplish both stages. R. Warrington had already seen a loss in nitrite oxidation capacity. Pure nitrite oxidizers were cultivated later in the twentieth century, however, it is impossible to know which cultures were free of impurities because all supposedly pure strains exhibit the same characteristic (nitrite consumption, nitrate production).

Nitrification and the Microbes

Ammonia-oxidizing bacteria (AOB) and ammonia-oxidizing archaea (AOA) are two types of organisms that convert ammonia to nitrite (AOA). Betaproteobacteria and Gammaproteobacteria are both home to AOB. Nitrosopumilus maritimus and Nitrososphaera Viennensis have been grown since the discovery of AOA in 2005. The Nitrosomonas and Nitrococcus genera are the most investigated AOB in soils. When AOB and AOA are compared in soils and marine habitats, AOA dominates, implying that Thaumarchaeota is a more important contributor to ammonia oxidation in these environments.

In the species Nitrospira inopinata, ammonia oxidation to nitrate in a single step was predicted in 2006 and discovered in 2015. In 2017, we were able to produce a pure culture of the organism, which marked a breakthrough in our knowledge of the nitrification process.

Nitrification and Nature

Both stages provide energy, which is then used to fuel ATP synthesis. Nitrifying organisms are chemoautotrophs, meaning they develop by consuming carbon dioxide. Urease is an enzyme that catalyzes the conversion of one urea molecule into two ammonia molecules and one carbon dioxide molecule in a certain AOB. Nitrosomonas Europaea, as well as colonies of soil-dwelling AOB, have been demonstrated to absorb carbon dioxide generated by the Calvin Cycle reaction to produce biomass and capture energy by oxidizing ammonia (the other result of urease) to nitrite. This property might explain why AOB grows faster in acidic settings when there is urea present.

In most situations, organisms can complete both phases of the process, resulting in nitrate as the final product. It is, nevertheless, feasible to build systems that produce nitrite. In agricultural settings, where fertilizer is frequently administered as ammonia, nitrification is critical. Because nitrate is more water-soluble than ammonia, converting it to nitrate enhances nitrogen leaching.

Nitrification is also an essential part of the nitrogen removal process in municipal wastewater. Nitrification is the most common method of elimination, followed by denitrification. Aeration (bringing oxygen into the reactor) and the addition of an external carbon source (e.g., methanol) for denitrification are the two most expensive parts of this process. Nitrification may happen in drinking water as well. The presence of free ammonia in distribution systems when chloramines are utilized as a secondary disinfectant can function as a substrate for ammonia-oxidizing microbes. As a result of the reactions, the disinfectant residual in the system may be depleted. It has been demonstrated that adding chlorite ions to chloramine-treated water controls nitrification.

Since nitrogen is frequently the limiting nutrient in the marine environment, the nitrogen cycle in the ocean is of great relevance. Because it produces nitrate, the principal form of nitrogen responsible for "new" generation, the nitrification stage of the cycle is of special relevance in the ocean. Furthermore, if the ocean gets increasingly enriched in human \[ CO_{2}\], the consequent drop in pH may result in slower nitrification rates. In the nitrogen cycle, nitrification might form a "bottleneck."

As previously stated, nitrification is a two-stage process in which ammonia is oxidized to nitrite in the first step and nitrite is oxidized to nitrate in the second. Each phase in the marine environment is controlled by a different bacterium. Nitrosomonas, Nitrospira, and Nitrosococcus are among the ammonia-oxidizing bacteria (AOB) found in the marine environment. All of them have the functioning gene ammonia monooxygenase (AMO), which is responsible for the oxidation of ammonia as its name suggests. Some Thaumarchaeota (previously Crenarchaeota) exhibit AMO, according to recent metagenomic research and culture methods.

Thaumarchaeota are ubiquitous in the ocean, and some species have a 200-fold higher affinity for ammonia than AOB, causing researchers to question the long-held view that AOB is the primary nitrifier. Furthermore, while nitrification is traditionally thought to be vertically separated from primary production because bacterial nitrate oxidation is inhibited by light, nitrification by AOA does not appear to be light inhibited, implying that nitrification is occurring throughout the water column, challenging the traditional definitions of "new" and "recycled" production. Some nitrification inhibitor examples include dicyanamide, nitrapyrin, and pronitradine.

Nitrite is oxidized to nitrate in the second phase. This phase is not as well understood in the seas as the first, although it is known to be carried out by the bacteria Nitrospira and Nitrobacter.

Nitrification and denitrification are also affected by the conditions and the nature of the soil. The soil conditions which control the nitrification rates are:

• The presence of \[NH _{4}^{+}\] indicates the availability of a nutrient

• Aeration is a term used to describe the process of \[(availability of O_{2})\]

• Soils that are well-drained and have a moisture content of 60%

• pH (potential hydrogen) (near neutral)

• Temperature (ideally 20-30 degrees Celsius) => Seasonal nitrification is influenced by land-use practices

In the end, nitrification, which is carried out by nitrifying bacteria, converts soil ammonia to nitrates (\[NO_{3}\]), which plants may take up and use in their tissues.