Nitrogen fixation

Importance of nitrogen fixation

Nitrogen accounts for 78.09% of atmospheric air. Above 1 hectare of land or water surface of the Earth there is about 80 thousand t of nitrogen, which is inaccessible to most higher plants.

Under natural conditions, when the level of mineral nitrogen in the soil is low, legumes are especially successful due to their ability to fix atmospheric nitrogen. A number of other plants possessing this feature, for example species of Gunnera (Gunnera), alder (Alnus) and Casuarina (Casuarina), have symbiotic relationships with soil-dwelling organisms that colonize their roots. Symbiosis of plants and beneficial organisms, as fossil data show, was one of the main factors in the success of plant spread. Although the genes required for signaling the onset of root colonization have been characterized, the origin of these genes remains unclear (Delaux et al., 2015).

Depending on energy sources, nitrogen-fixing microorganisms are classified as: autotrophs and heterotrophs.

Estimates put total annual nitrogen fixation in terrestrial ecosystems at 175–190 million t of nitrogen, 90–110 million t of which come from agricultural soils (Mishustin, 1983). At the same time, annual nitrogen removal from soil with agricultural products is 110 million t. According to other estimates, 50–140 million tons of nitrogen are fixed annually worldwide through nitrogen fixation (Unkovich et al., 2008). In 2009, about 0.8 million tons of nitrogen was fixed in the EU (Baddeley et al. (2013)).

Legumes have specific relationships with soil bacteria of the genus Rhizobium, in which bacteria penetrate the roots, leading to the process of converting atmospheric nitrogen into a soluble form. The ability to enter symbiosis with Rhizobium is one of the main reasons legumes are characterized as “pioneer plants,” since they usually colonize soils low in nitrogen and often in open environments (Cloutier et al., 1996). Crops capable of forming symbiotic relationships are one of the main sources of nitrogen in resource-limited agricultural systems.

After harvest, when most of the fixed nitrogen in the form of protein is removed from the field, residual nitrogen remains in the haulm, root system and nodules. Where roots remain to decompose in the soil, nitrate is released slowly and is available for uptake by the next crop. These levels can vary significantly, but are recognized to be about 20–50 kg N/ha (Sylvester-Bradley and Cross, 1991; Ward and Palmer, 2013). For faba beans, estimates made in the UK suggest fixation of 60–100 kg N/ha in stem and root residues after harvest (Iannetta et al., 2015).

This nitrogen source is economically valuable in a farming system, as it provides a nitrogen source immediately for the next crop. Because fixed nitrogen is released in the soil relatively slowly, the risk of leaching during wet periods is less than with the application of fertilizers, but, no less important, residual nitrogen available to the next crop reduces the need for it by about 50 kg/ha. In addition, the inclusion of a legume crop in crop rotation provides a “break” in a system based predominantly on cereals, reducing the accumulation of cereal pathogens such as the fungus Gaeumannomyces graminis, and providing an opportunity to control perennial weeds and grasses such as black-grass Alopecurus myosuroides, which has developed resistance to commonly used graminicides. In the EU it was estimated that the yield of winter wheat immediately after a legume crop increases by 0.6 and 0.9 t/ha (Von Richtofen, 2006), and a similar response has been noted in Canada (Wright, 1990). In some studies with faba beans (Vicia faba), including them in crop rotation increases the diversity of wild flora and fauna, as well as soil microflora (Kopke and Nemecek, 2010). Thus, there is convincing evidence in favor of field crop rotation supported by the inclusion of a legume crop (Iannetta et al., 2013).

Intensity of nitrogen fixation

The process of nitrogen fixation is carried out by free-living soil anaerobic bacteria Clostridium pasteurianum, aerobic Azotobacter chroococcum and nodule bacteria living in symbiosis on the root system of legumes, Rhizobium. Their activity and the efficiency of nitrogen fixation are influenced by the supply of carbohydrates, phosphorus, calcium and other elements, the reaction of the soil environment, temperature and moisture.

Efficiency is influenced by the nitrogen status of the soil. This is especially evident when the level of inorganic nitrogen in the soil is high and suppresses natural nodule formation and nitrogen fixation (Slattery et al., 2004). An experiment conducted by B. A. Yagodin together with Yu. Ya. Mazel and Yu. G. Sazonov in 1981 showed the dependence of symbiotic nitrogen fixation on plant nitrogen supply and photosynthesis intensity. In this experiment, lupine of the cultivar Bystrorastushchiy 4 was grown at different levels of nitrogen supply and under 1-, 3- and 6-day shading. Illumination changed 1000-fold. Shading led to decreased nitrogen fixation, to a greater extent at high mineral nitrogen content. After 6-day shading, nitrogen fixation in the no-nitrogen variant decreased 40-fold, in the half-dose variant it stopped completely, and at double doses nitrogen fixation stopped already after 3-day shading. The maximum intensity of nitrogen fixation was noted in the flowering phase in the no-nitrogen and half-dose variants. In the budding phase at half dose it was greater than in the no-nitrogen variant. This is explained by the fact that a small starter dose of nitrogen promotes better development of nodules at early stages of development. In the flowering phase in the no-nitrogen variant this indicator was higher than in the nitrogen variants. In the budding phase the maximum nitrogen fixation in the daily cycle occurred in the morning hours (8 h), and in the half-dose variant fixation proceeded faster than in the no-nitrogen variant. In the flowering phase the maximum occurred at noon. In this case it was greatest in the no-nitrogen variant. At increased nitrogen dose this indicator decreased in all developmental phases. More intensive influx of products labeled with ¹⁴C was noted in the no-nitrogen variant. At double dose it was 20% less. 30 min after exposure, the label was detected in the nodules of both variants (0.37 and 0.07 respectively, of total activity). Over 2.5 h, in the no-nitrogen variant 7 times more products entered the nodules than in the nitrogen variant, 5 times more into the roots, and 2 times more into the stems. The unequal rate of influx of photosynthesis products into root nodules at different levels of nitrogen nutrition influenced the intensity of nitrogen fixation. Due to the accumulation of photosynthesis products in the nitrogen variant, shading for 3 days suppressed nodule nitrogen fixation. Thus, shading of lupine leads to reduced nitrogen fixation, but against a background of mineral nitrogen this reduction is greater than without nitrogen.

The nitrogen fixation coefficient ranges from 0.3 to 0.85.

The intensity of nitrogen fixation by free-living bacteria depends on reserves of readily available organic substances serving as an energy source. For example, nitrogen fixation activity in the plant root zone due to associative nitrogen fixation is 3–200 times greater than in inter-row soils. Therefore, plants are the main factor in the activity of diazotrophic bacteria in the rhizosphere due to root exudation and root decay, the volume of which is from 25 to 50% of photosynthetic production.

The intensity of nitrogen fixation by diazotrophs is determined by the excretory activity of plant root systems, that is, ultimately by photosynthetic activity. High activity in the rhizosphere of many tropical plants is associated with the ability to use the C-4-dicarboxylic acid pathway in photosynthesis. Plants of this type require intense illumination, and their maximum photosynthesis rate is significantly higher than in plants using the Calvin cycle (C-3 type). Since C-4 plants consume fewer carbohydrates for photorespiration, part of them is used for root growth and root exudation.

Non-symbiotic nitrogen fixation has been studied by many researchers, but information about its scale in different soil-climatic zones is scarce, because under natural conditions this process depends on a number of dynamic environmental factors. Thus, according to several studies of fertile rice field soils, as a result of non-symbiotic fixation under rice, 60–70 kg/ha of nitrogen per year can accumulate. Moreover, in flooded soils 57–63 kg/ha of nitrogen is fixed, in non-flooded soils 3–7 kg/ha, and without plants in flooded soils 23–28 kg/ha of nitrogen.

Over 3 months of vegetation, nitrogen fixation in rice field soils of Krasnodar Krai was 9–27 kg. Incorporation of straw into the soil promotes the reproduction of various groups of nitrogen-fixing bacteria and an increase in nitrogen fixation to 20–40 kg/ha per month. Moisture also promotes increased activity during the decomposition of straw and cellulose. In intrazonal soils of excessive moisture, that is floodplain, bog soils and rice plantations, activity is highest — from 16.5 to 67.5 kg/ha per month. In soils of the tropical zone, non-symbiotic nitrogen fixation averages 200 kg/ha per year, sometimes reaching 600 kg/ha per year.

The activity of non-symbiotic nitrogen fixation also depends on: moisture, temperature, soil texture, degree of aeration of the root-inhabited layer, carbon dioxide content, and the presence of macro- and microelements. Mineral fertilizers, liming and air regime also influence intensity, but high efficiency is noted when moisture, temperature and organic matter do not limit nitrogen fixation. The addition of plant residues to sod-podzolic soils allows increasing nitrogen-fixing activity 2–5 times provided there is sufficient moisture.

Temperature influences the rate of nitrogen fixation, and extremely high or low temperatures reduce efficiency. Nitrogen fixation in legumes is sensitive to excess water (James and Crawford, 1998), drought and the level of soil salinity combined with high pH. Deficiency of key minerals can also limit nitrogen fixation, especially phosphorus. In alfalfa species (Medicago spp.) nodule size and weight decreased under P deficiency (Schultze and Drevon, 2005).

Mineral nitrogen fertilizers have a regulatory effect on nitrogen fixation. In in vitro experiments, inhibition of nitrogen fixation in the presence of combined nitrogen (in the absence of plants) was proven. Inhibition is noted at doses of combined nitrogen above 500–1000 kg/ha. Fertilizer doses used in agriculture cause short-term suppression of nitrogen fixation. At the same time, nitrogen fertilizers stimulate plant development, increase photosynthetic productivity and increase the scale of exudation, thereby promoting associative nitrogen fixation at later stages of plant development, when the excess mineral nitrogen of fertilizers in the soil significantly decreases.

The use of the radiometric method in studies of the amount of organic matter in stubble-root residues revealed that, due to root exudates during vegetation and the constant dying off of part of the root system after harvest, 3–4 times more organic matter remains in the soil than when determining this indicator by conventional methods. When calculating organic matter, aboveground plant organs that died during life should also be taken into account.

In symbiosis with nodule bacteria, legumes are able to satisfy up to 60–90% of their nitrogen requirement. Annual grain legumes, for example lupine, peas, bind 50–100 kg/ha of nitrogen per season, half of which remains in the soil; perennial forage legumes (clover, alfalfa) — 180–300 kg/ha, of which 70–100 kg/ha remains in the soil with roots and stubble residues.

Nitrogen-fixing bacteria

Autotrophic nitrogen fixers — cyanobacteria and photosynthetic anaerobic bacteria. They are significant only under conditions of excessive moisture and on flooded soils, where fixation reaches 20–50 kg/ha of nitrogen per year.

Heterotrophic nitrogen-fixing bacteria are distributed in all soil types in the rhizosphere and phyllosphere of plants. From this group, nodule bacteria and their role in the nitrogen nutrition of legumes and the enrichment of soils with nitrogen are well studied. The contribution of other heterotrophic symbiotic nitrogen fixers, for example endosymbionts of alder and sea buckthorn, is still poorly studied.

Non-symbiotic heterotrophic nitrogen-fixing bacteria are also little studied, although the nitrogen-fixing ability of microorganisms was discovered precisely in representatives of this group. In soils of the temperate climate zone they bind up to 3–5 kg/ha of nitrogen per year. Large species diversity and wide distribution have been revealed in soils of all climatic zones, in the rhizosphere and on plant surfaces, in water bodies, silts and sediments, in the intestines of insects, fish and higher animals.

The ability for nitrogen fixation has been found in a large number of bacteria of various systematic groups. Besides Azotobacter, Clostridium and nodule bacteria, bacteria Arthrobacter, Bacillus, Erwinia, Klebsiella and others possess this ability. Pure cultures of eukaryotic microorganisms, including fungi and yeasts, do not possess nitrogen-fixing activity, but mixed cultures of nitrogen fixers with eukaryotes have increased nitrogenase activity. Bacteria of the group Azotobacter develop well on aerated cultivated, well warmed, neutral soils containing phosphorus and calcium. Under favorable conditions they accumulate up to 30 kg of nitrogen per 1 ha.

Active nitrogen fixation by nodule bacteria is indicated by the pink or red color of nodules; when activity is low, nodules are white or pale green. To activate their activity, seeds of grain legumes are treated with bacterial fertilizers, for example rhizotorfin or nitragin.

Nodule bacteria are rods; in the free state they are strict aerobes and are not able to assimilate atmospheric nitrogen. Several species of nodule bacteria have been established, differing in the type of relationship to the host plant. Some bacterial species are able to infect a group of legumes, others are very specific and enter symbiosis only with individual crops.

Rhizobia (Rhizobium)

Not all legume species have a relationship with the same strain or species of soil-dwelling rhizobia bacteria (Rhizobium). Rhizobium organisms are free-living and motile in the soil and, as such, are not capable of fixing atmospheric nitrogen. Nodule formation will not occur if the corresponding species is not present in the soil. Within each Rhizobium species there are a number of strains specific to a particular host. Usually vetch (Vicia spp.) and pea (Pisum spp.) species are associated with Rhizobium leguminosarum, but it is believed that within this species different bacterial strains prefer each crop species. Recent studies have also shown that there are two different genetic classes of Rhizobium that infect faba beans (Vicia faba) (Del Egido, 2014). Bean (Phaseolus vulgaris) is associated with Rhizobium phaseoli; and lupine species, as another example, have a relationship with Bradyrhizobium lupini.

In many cases, the Rhizobium species/subspecies infecting specific host plants is present in the soil, but in some situations, especially when growing non-native crops, seeds may be inoculated with commercial preparations of specific strains or species of Rhizobium for that crop.

Rhizobium species and the range of their hosts (Smartt, 1976):

• Bradyrhizobium japonicum (syn. Rhizobium japonicum) — soybean (Glycine sp.);
• Rhizobium leguminosarum — pea (Pisum sp.), vetch (Vicia sp.), lentil (Lens sp.), grass pea (Lathyrus sp.);
• Bradyrhizobium lupini — lupine (Lupinus sp.);
• Ensifer meliloti (syn. Rhizobium meliloti) — sweet clover (Melilotus sp.), alfalfa (Medicago sp.), fenugreek (Trigonella sp.);
• Rhizobium phaseoli — bean (Phaseolus sp.);
• Rhizobium leguminosarum (syn. Rhizobium trifolii) — clover (Trifolium sp.).

The amount of fixed nitrogen depends on many factors, such as the effectiveness of the Rhizobium strain, the condition of the host plant, physical environmental factors, and agronomic practices. The amount of fixed nitrogen varies considerably; according to various data, it ranges from 45 kg to 550 kg N/ha for faba beans (Vicia faba) (Nutman, 1976) and was compared for a number of legume crop species in the work of Salunkhe and Deshpande (1991).

Relative nitrogen fixation by some legume crops (Salunkhe and Deshpande, 1991), in kg of nitrogen per 1 ha per year:

• alfalfa — 114–223, according to other data 250–300, sometimes up to 500;
• clover — 21–36 (150–160, sometimes up to 250);
• chickpea (ram) — 24–84;
• bean — 178–251;
• faba bean — 174–196;
• pea — 87–222;
• lentil — 167–189;
• lupine — 121–157 (160–170, sometimes up to 400);
• soybean — 22–310.

Associative nitrogen fixation

Associative nitrogen fixation — nitrogen fixation in the phytoplane, that is, the rhizosphere and phyllosphere of non-leguminous plants. For the first time, the possibility of nitrogen fixation in the root zone of non-leguminous crops was predicted in 1926 by S. P. Kostychev, subsequently experimentally confirmed by studies using the balance method (Broadbalk experiment (England), Pryanishnikov's experiment, the "perennial" rye experiment (Germany)).

Continuous cultivation of non-leguminous crops did not lead to a significant decrease in soil nitrogen content, despite its annual removal with the harvest, while in fallow the amount of nitrogen continuously decreased. In associative nitrogen fixation, microorganisms and plants do not enter into such an interaction as in symbiotic systems; however, on the whole, it has the same features: nitrogen fixation activity changes as plants develop, reaching a maximum during budding and flowering, and decreasing during ripening.

Initially, attention was paid to the interaction of nitrogen-fixing bacteria (Spirillum spp., Azospirillum lipoferum, Azospirillum brasilense, etc.) with the root system of tropical cereal plants. However, the claim about the uniqueness of the properties of azospirilla turned out to be exaggerated. At present, nitrogen fixation by the rhizosphere of rice, corn, sorghum, wheat, and some species of tropical grasses is known. More than 200 species of non-leguminous plants capable of nitrogen fixation with the help of rhizosphere microorganisms are known.

Associative nitrogen fixation is of great ecological importance; it is precisely through it that the fund of available nitrogen is replenished in most natural ecosystems. However, nodule bacteria (Rhizobium) in symbiosis with legume crops show the greatest effectiveness: under optimal conditions, biological nitrogen fixation reaches more than 300 kg/ha per year.

Mechanism of biological nitrogen fixation

At the end of the last century, various assumptions were made about the mechanisms of nitrogen fixation. Two variants were considered — oxidative and reductive. D. N. Pryanishnikov believed that the conversion of N₂ to NH₃ through nitrogen oxides was impossible, since it contradicted the principle of permissible energy economy of organisms. S. N. Vinogradsky at the end of the last century suggested a method of reductive fixation of molecular nitrogen to ammonia.

[.lightbox.float-right.w-350]

.Hypothetical scheme of nitrogen transformation during nitrogen fixation

image::scheme-of-nitrogen-transformation.jpg

The mechanism of the nitrogen assimilation process appears in this case as the action of hydrogen at the moment of its release on gaseous nitrogen in the living protoplasm of the cell. The hypothesis that ammonia synthesis is a direct result of this process seems to us well founded.

S. N. Vinogradsky On the assimilation of atmospheric gaseous nitrogen by microbes

P. A. Kostychev developed the theory of S. N. Vinogradsky on the reductive fixation of molecular nitrogen through ammonia. At present, this mechanism is considered generally accepted.

Nodule bacteria (Rhizobiales) are stimulated to infect through root hairs or through cracks in the epidermis of host plants by root exudates containing flavonoids, which are released into the soil during root growth (Sprent and James, 2007). The genetic mechanisms involved in initiating this infection are currently being studied. It has been shown that the presence of calcium and the associated signaling in root hairs is an important factor in the whole process. Nitrogen-fixing rhizobial bacteria that associate with legumes signal their hosts about the beginning of symbiosis by releasing diffusible compounds, including lipo-chito-oligosaccharides, also known as Nod factors. They cause calcium ion oscillations in root epidermal cells (Sun et al., 2015). The main elements of this signaling, being elucidated in current research, will become the cornerstone for studying the possibility of creating similar symbiotic associations with non-leguminous crops (Oldroyd and Dixon, 2014).

Having established themselves in the roots, the bacteria multiply and move into the root cortex along the infection thread, which is a tube made of cell walls. The roots respond to this infection by forming root nodules, which arise as a result of enlargement of root tissue cells. The movement of bacteria within the roots leads to the formation of new nodules, and as the nodules age they die off, releasing rhizobia into the soil for invasion into new roots. The nodules provide a specialized niche that allows rhizobia to fix nitrogen under conditions of minimal air quantity, which protects the enzyme nitrogenase but allows sufficient oxygen to be present for aerobic respiration associated with nitrogen fixation (Gallon, 1992; Minchin et al., 2008). The nodule also allows the symbiotic bacteria to efficiently exchange fixed nitrogen in return for carbohydrates from the host plant. Nitrogen is first converted to ammonia using energy obtained from carbohydrate metabolism (Emerich and Burris, 1978). Ammonia is then converted into amino acids, which are used by the bacteria to produce proteins and peptones necessary for their growth. The fixed nitrogen incorporated into rhizobia is then released in the form of amino acids, which are absorbed by the host plant (White et al., 2007; Anderson et al., 2013).

The main enzyme responsible for nitrogen fixation is nitrogenase. In legume crops it is concentrated in nodule bacteria. Difficulties in isolating this enzyme from cellular organisms long delayed the study of the biochemical processes of nitrogen fixation. Nitrogenase can be isolated only in the absence of air.

For the first time, cell-free extracts from soybean nodules were obtained in 1968 in Evans's laboratory. In the USSR, the enzyme complex (nitrogenase) was first isolated from bacteroids of lupine and soybean nodules in 1970.

Nitrogenase consists of two protein structures: one — with a molecular mass of 164,000, contains molybdenum and iron; the second — with a molecular mass of 56,000, contains only iron. Separately, these structures do not fix molecular nitrogen.

In most microorganisms, nitrogenase is inactivated by oxygen, and the Fe-protein is more sensitive to oxygen than the Mo-Fe-protein. The Fe-protein is also very sensitive to cold and is inactivated at a temperature of 0 °C. For the biochemical reaction of nitrogen reduction, the presence of ATP, an electron source, and Mg²⁺ ions is necessary. Most scientists believe that 15 molecules of ATP are consumed for the fixation of 1 molecule of nitrogen.

A distinctive feature of nitrogenase is its ability to reduce not only molecular nitrogen but also other molecules with a triple bond. This feature made it possible to use the method of determining nitrogen fixation by the reduction of acetylene (HC≡CH) to ethylene (H₂C=CH₂).

The biochemistry of nitrogenase has not been fully studied. For example, it is not known how the energy of ATP hydrolysis is coupled with electron transfer, how the reduction of the nitrogen molecule proceeds, and the mechanism of participation of iron and molybdenum in this process is unclear. The need for molybdenum in nitrogen-fixing Azotobacter is much greater than in nitrate-reducing species.

Other metal-containing enzymes also participate in symbiotic nitrogen fixation, for example, leghemoglobin, which is localized in plant cells. In nodules it is present as a product of symbiosis between bacteria and higher plants, but nitrogen fixation activity depends on its concentration in the nodules.

[.lightbox.float-right.w-350]

.Scheme of stepwise nitrogen reduction through diimide and hydrazine during nitrogen fixation (Hardy, 1970)

image::stepwise-nitrogen-reduction-scheme.jpg[Scheme of stepwise nitrogen reduction through diimide and hydrazine during nitrogen fixation]

Nitrogenase is very sensitive to oxygen, but rhizobia require oxygen for respiration. Nodules contain a high concentration of leghemoglobin, which transports oxygen and surrounds the bacteria in infected nodules. This leghemoglobin is capable of delivering oxygen to the bacteria at a low concentration, so its presence in nodules is necessary. Under anaerobic conditions or in waterlogged soils, the rate of nitrogen fixation decreases greatly, and a well-aerated soil type is necessary for efficient biological nitrogen fixation.

The function of leghemoglobin is similar to that of hemoglobin and consists in the reversible binding of oxygen. Leghemoglobin is an oxygen carrier; it does not participate in nitrogen reduction but participates in the processes of ATP energy formation, which proceed with the participation of oxygen, and at the same time allows anaerobic conditions to be maintained for nitrogenase operation. The mechanism of protecting nitrogenase from oxygen is complex, and leghemoglobin is probably only one link in this process.

Anaerobic nitrogen fixers presumably have several mechanisms for protecting nitrogenase from oxygen. For example, the very high respiratory activity of Azotobacter is a system of protection against inactivation by oxygen.

Another proposed method is "conformational protection of nitrogenase," that is, spatial change of the protein component of nitrogenase in the presence of oxygen.

Corrinoids — compounds of the vitamin B₁₂ group, including cobalt — participate in nitrogen fixation. The need for cobalt was shown in experiments on symbiotic cultivation of legumes on nutrient media devoid of nitrogen and was established by the fact of a positive effect on nitrogen fixation activity. In nodules, corrinoids are present in nodule bacteria (bacteroids) and in the plant tissue of the nodule.

The biochemical role of vitamin B₁₂ in nitrogen fixation is poorly studied; it is known that compounds of the B₁₂ group are part of the enzymes methylmalonyl-CoA mutase and ribonucleotide reductase.

Derivatives of vitamin B₁₂ catalyze biochemical reactions in which hydrogen is transferred between two adjacent carbon atoms with simultaneous movement of other groups in the opposite direction.

The role of cobamide coenzymes in the nodules of leguminous plants

Application of nitrogen fixation in agriculture

When solving practical problems related to nitrogen nutrition, an important place is occupied by the share of biological nitrogen in the nutrient balance. Data on the magnitude of symbiotic nitrogen fixation vary. For example, according to A. V. Sokolov, the amount of nitrogen fixed by clover varies from 45 to 95%; according to V. E. Shevchuk, only a third of nitrogen is fixed by legume plants from the atmosphere; according to E. N. Mishustin, per year in the root residues of alfalfa approximately 100 kg/ha of nitrogen accumulates and 50 kg/ha in clover.

According to calculations by N. S. Avdonin, based on data of D. N. Pryanishnikov on the ability of clover to fix 150 kg/ha of nitrogen per year, and alfalfa — 250–300 kg/ha, the magnitude of symbiotic assimilation of molecular nitrogen can reach 3 million t; according to calculations by E. N. Mishustin — about 3.5 million t. On the scale of the biosphere, the role of symbiotic nitrogen fixation is small, since legume crops account for about 10% of the total area of agricultural crop sowings, and in natural phytocenoses there are far fewer of them.

Under favorable conditions of symbiosis, that is, at pH_sol 6–7, provision with phosphorus, potassium, magnesium, boron, molybdenum, the presence of specific virulent strains of nodule bacteria, and optimal soil moisture, field pea fixes up to 150 kg/ha, faba beans and soybean — up to 250 kg/ha, white lupine — up to 300 kg/ha of nitrogen, while the yield is 3–4 t/ha of seeds. However, in practice it is rarely possible to ensure optimal conditions; the activity of symbiosis weakens and only 20–60 kg/ha of air nitrogen is fixed at a yield of 1.2–1.5 t/ha. Sometimes, due to excessive soil acidity, lack of moisture or nutrients, nitrogen fixation does not occur, and plants give low yields with a minimal protein content.

Data on nitrogen input into the soil with atmospheric precipitation are also insufficient. This value varies from 2 to 20 kg/ha per year. Up to 90% of the nitrogen supplied with seeds is used by the harvest. With seeds of grain legumes, from 8 to 15 kg/ha of nitrogen is applied; with cereal spike crops — 4–6 kg/ha.

The expenditure items of the nitrogen balance have been studied somewhat better. Numerous works by a number of authors have shown that fertilizer nitrogen on various soils is used by 50–60% in vegetation experiments and by 40–50% in field experiments. However, when determining nutrient utilization coefficients, the study of balanced plant nutrition with all necessary elements is extremely important.

According to V. K. Shilnikov and E. Ya. Serov, the reserve on a global scale of nitrogen fixation of terrestrial and aquatic ecosystems are blue-green algae and photosynthesizing diazotrophs. Some species of cyanobacteria form associations with fungi (lichens) and higher plants, for example, symbiotic associations with the floating fern Azolla (Azolla spp.). The effectiveness of many aerobic nitrogen-fixing microorganisms increases at an oxygen pressure of less than 2.02⋅10⁴ Pa. Therefore, in sea and ground water, in soils of flooded rice fields, and in hot springs, favorable conditions for nitrogen fixation by aerobic microorganisms may develop.

The phyllosphere, that is, the surface of plant leaves, also serves as a niche for non-symbiotic heterotrophic binding of atmospheric nitrogen by microorganisms. Its contribution to the overall nitrogen accumulation balance is estimated at 13–15%, and the nitrogen-fixing capacity of phyllosphere microorganisms at 55%.

D. N. Pryanishnikov paid great attention to the nitrogen balance as a criterion of soil fertility and crop yield. He was the first to calculate the nitrogen balance in the USSR. In 1937, the nitrogen deficit was about 70%. According to his calculations, in 1940 most of the nitrogen returned to the land came: with manure — up to 14.8%, with legume roots — 8.2%, with mineral fertilizers — 3.2%, total — 26.2%. The remaining 73.8% was the deficit.

The study of the processes of microbiological nitrogen fixation will make it possible to use it more effectively in agriculture.

The most important practical issues of biological nitrogen that require study include:

1. Ecological-biological and agronomic aspects of the process for full use and intensification of nitrogen fixation.

2. Study of the mechanisms of biological binding under mild conditions for developing new methods of producing nitrogen fertilizers and techniques for regulating nitrogen fixation processes.

3. Genetic-selection studies of the symbiosis of leguminous plants with nodule bacteria, as well as the application of genetic engineering, achievements of biochemistry and molecular biology for extending nitrogen fixation processes to many agricultural crops.

References

1. Yagodin B. A., Zhukov Yu. P., Kobzarenko V. I. Agrochemistry. / Ed. B. A. Yagodin. M.: Kolos. 2002.

2. Vavilov P. P., Gritsenko V. V., Kuznetsov V. S. et al. Crop Production / Ed. Vavilov P. P. 5th ed., revised and enlarged. M.: Agropromizdat 1986