Nitrifying bacteria (ammonium oxidizing bacteria -AOB-, and nitrite oxidizing bacteria -NOB-): Quantification by real - time PCR.  

Information 2003-02-16..

Nitrifying bacteria are chemoautotrophic bacteria (synthesized alone all the chemical components of structures from simple chemical elements), or chemolithotrophic (synthesized all chemical components of structures from inorganic compounds, and obtain energy during this process) . These bacteria are ubiquitous in nature, being found in the soil, lakes and streams rivers with high content in wastewater. Its importance is involved in the nitrogen cycle in nature.

Nitrogen can be free in the atmosphere (N _2), or part of the organic matter, both amino acids (amino groups -NH ₂ -) and nucleic acids (DNA and RNA) (nucleotide purine and pyrimidine bases nitrogenous). It is one of the indispensable elements of organic matter, nitrogen and if there were not exist life.

Nitrogen can reach the ground through the ammonia _(NH3) from organic matter of living things (droppings, plant detritus, animal carcasses, ...,). There may also be nitrogen in the soil where there are bacteria capable of fixing atmospheric nitrogen (N _2), turning it into ammonium (NH ₄ ^+). Several species of bacteria capable of fixing nitrogen, some associated with the roots of leguminous plants (peas, ....) such as Rhizobium spp., And others that are free as with Azotobacter spp., Beijernickia spp. Clostridium spp., Cyanobacteria spp., Etc. The process by which fixing bacteria atmospheric nitrogen incorporate nitrogen to the soil as ammonia is called Ammonification. One of the most important bacteria in this process is Azotobacter spp.

The ammonia that is found in soil and can oxidize converted into nitrites and these in turn then converted into nitrates. The process of conversion of ammonia to nitrite and nitrate is called nitrification. Nitrification is a process in two stages. The first ammonium (NH ₄ ⁺⁾ is oxidized to nitrite by oxidizing bacteria ammonium (AOB: Ammonium oxidazing bacteria) and in the second, nitrites are oxidized to nitrate by oxidizing bacteria nitrites (NOB: Nitrite oxidazing bacteria). Subsequently, nitrates can be converted back into free nitrogen by denitrifying bacteria, called denitrification process. Denitrifiers are many gram - negative bacteria facultative anaerobes. Enterobacteriaceae, Pseudomonas spp, Bacillus licheniformis, Paracoccus spp, and even one of the nitrifying bacteria, Nitrosococcus spp.. since this bacterium is involved in both the nitrification and denitrification in. Thus nitrogen atmosphere again completing the cycle.

The nitrification process is carried out by two groups of chemolithotrophic bacteria (bacteria obtain energy using inorganic chemical compounds), phylogenetically unrelated.

Nitrifying bacteria were initially included in the Nitrobacteraceae family. When the ribosomal ribonucleic acid (rRNA 16S) was sequenced, the family was divided as it was found that included genetically diverse bacteria. Currently nitrifying bacteria are classified in the following genera:

Nitrite formation from ammonium (nitrification - first stage) by oxidizing bacteria ammonium possessing mono-oxygenase ammonium (AMO) and hydroxylamine-oxidoreductase (HAO: hidroxilamine oxidoreductase):

• • • Nitrosomonas spp. (Class betaproteobacterias).
    • Nitrosospira spp. (Class betaproteobacterias).
    • Nitrosococcus spp. (Gamma - Proteobacteria class).

Transformation of nitrites to nitrates (nitrification - second stage), by nitrite - oxidizing bacteria, nitrite having oxidoreductase (NXR):

• • • Nitrobacter spp. (Alfaproteobacterias Class).
    • Nitrospira spp. (Deltaproteobacterias Class).
    • Nitrococcus spp. (Gamma - Proteobacteria class).

The microbiological characteristics of the main nitrifying bacteria are as follows:

• • • Nitrosomonas spp. (N. europea): short or long, mobile or immobile gram - negative bacilli, found on the floor (ground), sewage, freshwater or marine waters.

• • • Nitrosospira spp .: spirals, peritrichous flagella for motile bacteria which are in the ground (earth).

• • • Nitrosococcus spp. (N. Oce; N. halophilus; N. nitrosus; N. vatsoni) gramnegative bacteria spherical or ellipsoidal, in pairs or tetrads, mobile, found in fresh or marine waters. These bacteria besides participating in the nitrogen cycle, also involved in the sulfur cycle, reducing sulphates to SH _2. Denitrifying also have activity releasing nitrogen into the atmosphere from the nitrates.

• • • Nitrobacter spp. Short, mobile or immobile, grannegativos bacillus. They inhabit the soil (earth), freshwater or marine waters.

• • • Nitrospira spp. (N. gracilis; marine N.). Helical or as vibrio (coma) bacteria. They are on the ground (earth) and in marine waters.

• • • Nitrococcus spp. (N. mobilis). Aerobic gram - negative cocci mobile I halófilo must present only in seawater as it is a must halófilo. It is more sensitive to light than Nitrobacter spp. and therefore do not live near the sea surface, nor very deep because it requires oxygen. This bacterium is very important in the nitrogen cycle in marine environments. Also involved in the marine carbon cycle fixing carbon which is then used by heterotrophic bacteria.

The ammonium oxidation is a complex process that requires two enzymes: ammonium-mono-oxygenase (AMO) and hydroxylamine-oxidoreductase (HAO) because initially the ammonium becomes hydroxylamine (NH ₂ OH) (catalyzed by ammonium-mono -oxigenasa), and posteriormenmte, it becomes nitrites (HNO ₂₎ (catalysed by the oxidoreductase hydroxylamine).

• • • NH ₃ + 1½ O ₂ -> HNO ₂ + H ₂ O (Nitrite formation: ammonium mono-oxygenase and hydroxylamine oxidoreductase)

• • • HNO ₂ + H ₂ O -> HNO ₃ (nitrate formation: nitritooxidorreductasa)

Subsequently, other bacteria such as Nitrobacter spp. nitrites to nitrates oxidized using oxygen as a final electron acceptor. Thus the final product of the bacteria involved in the first stage, as is Nitrosomonas spp., Is the energy substrate for bacteria of the second stage, as is Nitrobacter spp. For this reason, both groups of bacteria are usually associated in nature.

Its development is harmful light, which in nature are covered with a biofilm, and laboratory cultures should be protected from light.

In fertilizers, for example, must be abundant Azotobacter spp. to fix atmospheric nitrogen (ammonification), and there should be the least amount possible oxidizing nitrifying bacteria ammonium (AOB) to prevent transform ammonium into nitrite, and this in turn is converted by oxidizing bacteria nitrites (NOB) nitrate .

In agriculture, the bacteria that perform the first stage of nitrification, as Nitrosomonas spp., Are a problem because convert ammonium, used as fertilizer, nitrites, and it can not be used by plants as ammonium. Nitrites can also contaminate groundwater being harmful for consumption.

These bacteria may also live in the walls of buildings and other surfaces (eg monuments) of polluted areas in which the atmosphere contains copious amounts of nitrogen compounds. When using ammonium produce nitric acid, and this acid dissolves some stones and other building materials of statues and buildings. You can reach even 15 cm penetrate deep into the sandy stones.

These bacteria have a low growth rate, are sensitive to many environmental factors such as the ammonium concentration, the oxygen concentration, the intensity of light, nutrients, pH, salinity, temperature changes, and are unable to compete with heterotrophic bacteria (bacteria obtain energy by metabolizing organic compounds). The difficulties of culturing in vitro these bacteria has limited its quantification methods as the most probable number (MPN). For this reason, the quantification methods based on PCR have found their applications in this area.

The development of molecular methods for detecting the 16S rRNA gene (16S ribosomal ribonucleic acid) and amoA (ammonium oxidizing enzyme) gene allowed the study populations ammonium oxidizing bacteria in various types of environmental conditions. Have used different molecular methods such as fluorescence in situ using fluorescent probes (FISH), amplification with specific PCR, methods of quantitative PCR such as PCR limiting dilution, kinetic PCR, competitive PCR, and real time PCR (Real-time PCR).

Tests in IVAMI

• Quantification of all nitrifying bacteria present in a sample per unit weight or volume and quantification of ammonium oxidizing bacteria (AOB) by real - time PCR.