Nitrification

NITRIFICATION, biochemical process of oxidation of ammonia into nitric acid, the causative agents of which are nitrifying bacteria. Pure cultures of these bacteria were first obtained in 1889 by Winogradsky, and his subsequent research laid a firm foundation for the modern understanding of this phenomenon. Experiments with pure cultures of the causative agents of nitrification showed that it proceeds in two phases and is caused by the life activity of two groups of nitrifying bacteria. In the first phase, nitrite bacteria (Bacillus nitrosomonas and Bacillus nitrosococcus) oxidize ammonia to nitrous acid; the second phase of nitrification, the oxidation of nitrous acid to nitric acid, is the result of the life activity of nitrate bacteria (Bacillus nitrobacter). As they form, nitrous and nitric acids are usually bound by bases present in the environment and form salts of these acids: nitrites and nitrates. Winogradsky described several species of nitrite bacteria. Representatives of Nitrosomonas are distributed exclusively in the soils of the Old World. These are oval rods (Figures 1 and 2) 0.8 - 1 µ x 1.2 - 1.8 µ, aerobes, Gram-positive and staining well with ordinary aniline dyes. In liquid media, the microbe grows in the first days into zoogloeae, which, as ammonia is consumed, break down into separate motile cells equipped with a single flagellum (monad stage). In this stage, Nitrosomonas rapidly completes the oxidation of ammonia, after which the motile cells drop and cover the bottom of the flask with a dense zoogloea. Varieties of Nitrosococcus are distributed mainly in the soils of America and Australia. They are characterized by a spherical shape (cocci). Cell dimensions of various varieties vary from 0.6 µ to 2 µ in diameter. Gram-positive and easily stained with ordinary aniline dyes; non-motile. In liquid media, they grow as separate cells. Most varieties do not form zoogloeae. Nitrate bacteria isolated from different places turned out to be completely identical. Only one species, Bacillus nitrobacter, has been described (1891), representing a small, non-motile, aerobic rod 0.3 - 0.4 µ x 1 µ (Figure 3); poorly stained; stains with carbol fuchsin only upon heating. To obtain cultures of nitrifying bacteria and to study nitrification under laboratory conditions, Winogradsky proposed elective mineral media.

Figure 1. Bacillus nitrosomonas. Monad stage.

For experiments with nitrite bacteria, a mineral solution containing some ammonium salt is inoculated with a small lump of earth or a drop of sewage. The course of nitrification is judged by the appearance of nitrous acid (Griess reagent) and by the disappearance of ammonia (Nessler reagent). For the isolation of individual colonies of nitrite bacteria, solid mineral media are convenient: silica gel and magnesium-gypsum plates. The elective medium for nitrate bacteria is a mineral solution containing some salt of nitrous acid. It is inoculated with a lump of earth or a drop of sewage. As nitrate bacteria develop, the nitrite disappears (Griess reagent), oxidizing to nitrate (diphenylamine test). The solid medium for isolating individual colonies of Bacillus nitrobacter is nitrite agar. The growth of nitrifying bacteria on media not containing organic compounds indicates their physiological characteristics: they are typical autotrophic organisms. For their development, they do not need ready-made organic matter, because they synthesize it from CO₂ at the expense of thermal energy released during oxidation reactions. Thus, during the oxidation of ammonia to nitrous acid and the latter to nitric acid, the following amount of calories is released: in the 1st phase (2NH₃+3O₂=2HNO₂+2H₂O), 158 calories are released; in the 2nd phase (2HNO₂+O₂=2HNO₃), 43.2 calories are released. In accordance with the thermochemical effect of oxidation reactions, the ratio of oxidized nitrogen to assimilated carbon for nitrite and nitrate bacteria is far from identical.

Nitrobacter experiments with nitrite bacteria

Figure 3. Bacillus nitrobacter.

Winogradsky's experiments established that cultures of Bacillus nitrosomonas assimilate 1 atom of carbon at the expense of oxidizing an average of 35 molecules of ammonium nitrogen: Oxidized N... 722.0

506.1

928.3

15.2

26.4

33.3

35.2

36.4 Similar experiments by Meyerhof showed that in cultures of Bacillus nitrobacter, 1 atom of carbon is assimilated at the expense of oxidizing an average of 135 molecules of nitrite nitrogen: Oxidized N... 475

3.55

2.63

2.95 Ratio N:C. 135

128 The cited figures show that nitrifying bacteria obtain the necessary amount of carbon at the cost of greater oxidative work, which explains the extremely slow growth characteristic of their cultures. The second physiological feature of nitrifying bacteria is their negative attitude towards organic compounds. The introduction of glucose, peptone, etc., into the nutrient solution sharply suppresses the development and oxidative activity of nitrifying bacteria. It should be noted that nitrate bacteria are less sensitive to organic matter than nitrite bacteria. The good development of nitrate bacteria on nitrite agar already indicates this property. The following table shows the doses of organic substances (in %) that delay and stop the development of nitrifying bacteria. But while being more resistant to organic matter, nitrate bacteria react sharply to the presence of ammonia. The content of 0.0005% ammonia in the nutrient solution suppresses the development of Bacillus nitrobacter. Bacterial species | Glucose | Peptone | Asparagine Nitrite bacteria: Delays development | 0.025 | 0.025 | 0.025 Stops development | 0.05 | 0.2 | 0.3 Nitrate bacteria: Delays development | 0.05 | 6.8 | 0.05 Stops development | 0.2 | 1.25 | 0.5 Under natural conditions, both groups of nitrifying bacteria are always found together, forming an obligatory symbiosis. Nitrite bacteria, by oxidizing ammonia, relieve nitrate bacteria from a compound so harmful to them, converting it into energy material necessary for their development—nitrite. Nitrate bacteria, in turn, by oxidizing nitrous acid to nitric acid, relieve nitrite bacteria from the waste products of their life activity. Both phases of nitrification proceed simultaneously, and the intermediate product—nitrites—cannot in most cases be accounted for. It should also be noted that under natural conditions, nitrification proceeds even in the presence of organic matter. In nature, nitrifying bacteria are extremely widely distributed. The simplicity of their metabolism allows them to settle where there is yet no life; they are found on bare rocks, in reservoirs, and in the upper layers of the earth's crust; soil is their main habitat. In cultivated soils, nitrification proceeds particularly intensively, because the cultivation of the arable layer increases the access of atmospheric oxygen necessary for oxidation reactions. The significance of nitrification in nature fully corresponds to the wide distribution of its causative agents. Ammonium salts formed during the decomposition of proteins and urea are converted by the activity of nitrifying bacteria into nitrates, which are the best source of nitrogen for plants. Thus, nitrification closes the nitrogen cycle in nature. The power of the nitrification process can be judged by the fact that the famous deposits of nitric acid salts (saltpeter) in Chile were formed by the activity of nitrifying bacteria. With proper cultivation in a podzolic soil resting under black fallow, as a result of the work of nitrifying bacteria during the summer months, up to 800-1120 kg of nitric acid salts per hectare can accumulate (S. P. Kravkov). In chernozems under the same conditions, even more nitrates are formed. The significance of nitrifying bacteria in sanitary engineering is no less great. During wastewater purification at treatment plants of all types, whether irrigation fields, filtration fields, purification ponds, or various biological oxidation plants (see Biological method of sewage purification), nitrification begins everywhere following the partial oxidation of organic matter. The presence of nitrates in purified waters increases their relative stability (ensures non-putrescibility) due to the oxygen bound in nitrates (see Denitrification). Therefore, one of the main tasks of wastewater purification is the creation of the best conditions for nitrification. The high rate of mineralization of organic matter during wastewater purification by aeration with activated sludge is due to the fact that the introduction of activated sludge enriches the sewage with nitrifying bacteria along with microbes that oxidize organic matter, and the artificial introduction of air provides the oxygen necessary for their oxidative activity. Temperature and the reaction of the medium also have a great influence on the rate of nitrification. According to Bazyakina's observations, at 9–26° the rate of nitrification is the same, but at 6° the process sharply slows down and at 0° does not go at all. According to Meyerhof, the optimal reaction of the medium for nitrite bacteria lies between pH = 8.4–8.8. For nitrate bacteria, the optimal reaction lies between pH = 8.3–9.3.