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Nitrogen fixation

Nitrogen fixation, or microbial fixation of atmospheric nitrogen, is the process of absorption of atmospheric nitrogen by soil microorganisms and its transformation into organic and mineral substances.

Nitrogen fixation was studied by J. Boussengo, M. Beiernik, H. Gelriegel, H. Wilfort, M.S. Voronin, S.N. Vinogradsky, V.L. Omelyansky, D.N. Pryanishnikov, D.I. Mendeleev, K.A. Timiryazev.

“There are few phenomena where the mutual role of theory and practice would be so clearly defined as in those studies in which scientific questions about the origin of nitrogen in plants were inseparably merged with purely practical questions about the usefulness of cultivating clover and legumes in general.”

K.A. Timiryazev, 1890.

Russian agricultural science paid great attention to the study of the phenomenon of nitrogen fixation: a collection of the most effective strains of microorganisms was created, genetic and genetic-breeding studies have been carried out since the late 50s, which were first covered in the Russian literature in the monograph “Genetics of Symbiotic Nitrogen Fixation with Selection Basics”, edited by I.A. Tikhonovich and N.A. Provorov in 1998.

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The 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 tons of nitrogen, which is inaccessible to most of the higher plants.

Nitrogen atoms in the N2 molecule are connected by a very strong triple bond N≡N, so breaking this bond is associated with high energy costs. In industry, this process with the formation of ammonia occurs at high temperatures and pressure, whereas in biological systems – at normal atmospheric pressure and temperature.

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

The total annual volume of nitrogen fixation in terrestrial ecosystems is estimated at 175-190 million tons of nitrogen, 90-110 million tons of which come from farmland soils (Mishustin, 1983). At the same time, the annual removal of nitrogen from soils with agricultural products is 110 million tons.

Nitrogen fixation intensity

The experiment conducted by B.A. Yagodin together with Y.Y. Mazel and Y.G. Sazonov in 1981 showed the dependence of symbiotic nitrogen fixation on plant nitrogen availability and photosynthesis intensity.In this experiment, lupine variety Bystrorastushchiy 4 was grown under different levels of nitrogen supply and 1, 3 and 6-day shading. Illumination levels varied by a factor of 1,000. Shading led to a decrease in nitrogen fixation, more so at high mineral nitrogen content. After 6-day shading, nitrogen fixation in the variant without nitrogen decreased 40-fold, in the variant with a half dose – completely stopped, at double doses – nitrogen fixation stopped already after 3-day shading.

The maximum intensity of nitrogen fixation was observed in the flowering phase in the variants without nitrogen and at half dose. In the phase of budding with a half dose it was more than in the variant without nitrogen. This is explained by the fact that a small starting dose of nitrogen promotes better development of nodules in the early stages of development. In the phase of flowering in the variant without nitrogen this index was higher than in the variants with nitrogen.

In the phase of budding, the maximum nitrogen fixation in the daily cycle occurred in the morning hours (8 h), and in the variant with a half dose, fixation was faster than in the variant without nitrogen. In the flowering phase, the maximum was at noon. In this case it was the highest in the variant without nitrogen. With an increased dose of nitrogen, this index decreased in all phases of development.

More intense intake of 14C-labeled photosynthetic products was observed in the variant without nitrogen. At the double dose, it was 20% less. After 30 min of exposure, the label was detected in nodules of both variants (0,37 and 0,07, respectively, of the total activity). Within 2.5 h, the nitrogen-free variant had 7 times more products in the nodules than the variant with nitrogen, 5 times more in the roots, and 2 times more in the stems.

The unequal rate of photosynthesis products entering the root nodules at different levels of nitrogen nutrition influenced the intensity of nitrogen fixation. Due to the accumulation of photosynthetic products in the variant with nitrogen, shading for 3 days suppressed nitrogen fixation in nodules.

Thus, lupine shading leads to a decrease in nitrogen fixation, but in the variant with a background of mineral nitrogen this reduction is greater than without nitrogen.

Nitrogen fixation coefficient ranges from 0.3 to 0.85.

The intensity of nitrogen fixation by free-living bacteria depends on the stock of readily available organic substances serving as a source of energy. For example, the nitrogen fixation activity in the root zone of plants due to associative nitrogen fixation is 3-200 times higher than in soils between the rows. Therefore, plants are the main factor of diazotrophic bacteria activity in rhizosphere due to root exudation and root fall, the volume of which is 25 to 50% of photosynthetic production.

The intensity of nitrogen fixation by diazotrophs is determined by the excretory activity of plant root systems, i.e., 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 light, and their maximum photosynthetic rate is much higher than that of plants using the Calvin cycle (C-3-type). Since C-4-type plants use less carbohydrates for photorespiration, some of them are used for root growth and root exudation.

Nonsymbiotic nitrogen fixation has been studied by many researchers, but there is little information about its extent in different soil and climatic zones, due to the fact that under natural conditions, this process depends on a number of dynamic environmental factors.

Thus, according to a number of studies of fertile soils of rice fields, it is shown that non-symbiotic fixation under rice accumulates 60-70 kg/ha of nitrogen per year. Moreover, 57-63 kg/ha of nitrogen is fixed in flooded soils and 3-7 kg/ha in non-flooded soils; without plants in flooded soils, 23-28 kg/ha of nitrogen is fixed.

During 3 months of vegetation, nitrogen fixation in soils of rice fields in Krasnodar region of Russia was 9-27 kg/ha. Introduction of straw into the soil promotes the multiplication of different groups of nitrogen-fixing bacteria and increases nitrogen fixation to 20-40 kg/ha per month. Humidity also contributes to increased activity in straw and cellulose decomposition. In intrazonal soils of excess moisture, i.e. floodplain, marsh soils and rice plantations, activity is highest, ranging from 16.5 to 67.5 kg/ha per month. In tropical soils, 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, granulometric soil composition, degree of aerated root layer, carbon dioxide content, availability of macro- and microelements. Mineral fertilizers, liming, air regime also affect the intensity, but, high efficiency is noted when moisture, temperature and organic matter do not limit nitrogen fixation. Application of plant residues to sod-podzolic soils allows increasing nitrogen-fixing activity by 2-5 times if there is sufficient moisture.

Mineral nitrogen fertilizers have a regulating effect on nitrogen fixation. In in vitro experiments inhibition of nitrogen fixation in the presence of bound nitrogen (in the absence of plants) has been proved.

Inhibition is observed at doses of bound nitrogen greater than 500-1000 kg/ha. Fertilizer doses, which are generally used in farming, cause short-term inhibition of nitrogen fixation. At the same time, nitrogen fertilizers stimulate plant development, increase photosynthetic productivity and increase the scale of exoosmosis, thus contributing to associative nitrogen fixation at later stages of plant development, when the excess of fertilizer mineral nitrogen in the soil is significantly reduced.

Application of radiometric method in the study of the amount of organic matter in stubble and root residues revealed that at the expense of root excretions during the growing season and the constant dying off of the root system after harvesting plants in the soil remains 3-4 times more organic matter than in determining this indicator by conventional methods. When calculating organic matter, we should also take into account the dead above-ground organs of plants.

In symbiosis with nodule bacteria, legumes can meet up to 60-90% of their nitrogen demand. Annual leguminous crops, such as lupine, peas, for a season bind 50-100 kg / ha of nitrogen, half of which remains in the soil, perennial legumes (clover, alfalfa) – 180-300 kg / ha, of which with roots and crop residues remains in the soil 70-100 kg / ha.

Nitrogen-fixing bacteria

Autotrophic nitrogen fixers are cyanobacteria and photosynthesizing anaerobic bacteria. They are important only in overmoistened conditions and on flooded soils, where nitrogen fixation reaches 20-50 kg/ha per year.

Heterotrophic nitrogen-fixing bacteria are distributed on all types of soils in rhizosphere and phyllosphere of plants. Among this group, nodule bacteria and their role in nitrogen nutrition of legumes and nitrogen enrichment of soils are well studied. The contribution of other heterotrophic symbiotic nitrogen fixers, such as alder and buckthorn endosymbionts, is still poorly studied.

Non-symbiotic heterotrophic nitrogen-fixing bacteria are also poorly studied, although nitrogen-fixing ability of microorganisms has been discovered just in representatives of this group. In soils of temperate zone they fix up to 3-5 kg/ha of nitrogen per year. A high species diversity and their wide distribution in soils of all climatic zones, in the rhizosphere and on the surface of plants, in water bodies, muds and sediments, in the intestines of insects, fish and higher animals have been revealed.

The ability to nitrogen fixation was found in a large number of bacteria of different systematic groups. In addition to Azotobacter, Clostridium and nodule bacteria, the following groups of bacteria possess this ability: Arthrobacter, Bacillus, Erwinia, Klebsiella, etc. Nitrogen-fixing activity is absent in pure cultures of eukaryotic microorganisms, including fungi and yeasts, but mixed cultures of nitrogen-fixers with eukaryotes are distinguished by the increased nitrogenase activity.

The active activity of nodule bacteria is determined by pink or red coloring of nodules, while slightly active nodules are white or pale green. To activate their activity, the seeds of legume crops are treated with bacterial fertilizers, such as rhizotorfin or nitragin.

Nodule bacteria are bacilli (rod); in the free state, they are strict aerobes and are unable to assimilate air nitrogen. Several species of nodule bacteria have been identified, differing in the type of relationship to the host plant. Some species of bacteria are capable of infecting a group of leguminous plants, while others are very specific and symbiotic only with individual crops.

Associative nitrogen fixation

Associative nitrogen fixation is nitrogen fixation in the phytoplanus, i.e. rhizosphere and phyllosphere of nonlegumes. For the first time, the possibility of nitrogen fixation in the root zone of nonlegume crops was predicted in 1926 by S.P. Kostychev and later experimentally confirmed by studies using the balance method (the Bradbock experiment (England), the Pryanishnikov experiment, the “eternal” rye (Germany)).

Permanent cultivation of nonlegume crops did not lead to a significant decrease in the content of nitrogen in the soil, despite its annual alienation with the harvest, at the same time, the amount of nitrogen in steam was continuously decreasing. In associative nitrogen fixation, microorganisms and plants do not enter into the same interaction as in symbiotic systems, but, in general, it has the same features: nitrogen fixation activity changes with plant development, the maximum is reached during budding and flowering periods, decreasing at ripening.

Initially, attention was paid to the interaction of nitrogen-fixing bacteria (Spirillum, Lipoferum, Azospirillum brasilense, etc.) with the root system of tropical cereal plants. However, the statement about the uniqueness of the properties of Azospirillum has proven to be exaggerated. At present, nitrogen fixation by rhizosphere of rice, corn, sorghum, wheat, and some species of tropical grasses is known. More than 200 species of nonlegumes are known to be capable of nitrogen fixation by rhizosphere microorganisms.

Associative nitrogen fixation is of great ecological importance; it is at the expense of 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 efficiency: under optimal conditions, biological nitrogen fixation reaches more than 300 kg/ha per year.

Mechanism of biological nitrogen binding

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

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

S.N. Vinogradsky “On the assimilation of gaseous nitrogen of the atmosphere by microbes”.

Hypothetical scheme of nitrogen transformation during nitrogen fixation
Hypothetical scheme of nitrogen transformation during nitrogen fixation

P.A. Kostychev developed S.N. Vinogradsky’s theory of reductive fixation of molecular nitrogen to ammonia. Currently, this mechanism is considered universally accepted.

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 have long delayed the study of biochemical processes of nitrogen fixation. Nitrogenase can be isolated only in the absence of air.

Cell-free extracts from soybean nodules were first obtained in 1968 in the Evans laboratory. In the USSR, an enzyme complex (nitrogenase) was isolated for the first time from bacteroides of lupine and soybean nodules in 1970.

Nitrogenase consists of two protein structures: one, with a molecular weight of 164,000, contains molybdenum and iron; the other, with a molecular weight of 56,000, contains only iron. Individually, 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. Fe-protein is also very sensitive to cold and is inactivated at 0 °C. The biochemical reaction of nitrogen reduction requires ATP, an electron source, and Mg2+ ions. Most scientists believe that it takes 15 molecules of ATP to fix 1 molecule of nitrogen.

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

The biochemistry of nitrogenase is not fully understood. For example, it is not known how the energy of ATP hydrolysis combines with electron transfer, how the reduction of the nitrogen molecule proceeds, and the mechanism of iron and molybdenum participation in this process is not clear. The molybdenum requirement of nitrogen-fixing Azotobacter is much greater than that of nitrate-reducing species.

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

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

The function of leghemoglobin is similar to that of hemoglobin and consists in the reversible addition of oxygen. Leghemoglobin is an oxygen carrier and does not take part in nitrogen reduction, but participates in the processes of ATP energy formation, which take place with the participation of oxygen, and simultaneously allows to maintain anaerobic conditions for nitrogenase operation. The mechanism of nitrogenase protection from oxygen is complex, and leghemoglobin is probably only one of the links in this process.

Anaerobic nitrogen fixers presumably have several mechanisms to protect nitrogenase from oxygen. For example, the very high respiratory activity of Azotobacter is a defense system against oxygen inactivation.

Another putative mechanism is the “conformational protection of nitrogenase,” that is, the spatial change of the protein component of nitrogenase in the presence of oxygen.

Corrinoids, compounds of the vitamin B12 group that include cobalt, take part in nitrogen fixation. The need for cobalt was shown in experiments on symbiotic cultivation of legumes on nutrient media deprived of nitrogen and established by the fact of positive effect on nitrogen fixation activity. In nodules, corrinoids are present in nodule bacteria (bacteroides) and in the plant tissue of the nodule.

Таблица. Content in nodules of substances involved in nitrogen fixation

Substances
Effective strain
Ineffective strain
Vitamin B12 in lupin nodules, mmkg/g
65
45
Cobamide coenzymes in cells Rhizobium melitoli, thousand mmol/g
25.5
6.7
Hemoglobin in bean nodules (according to Shemakhanova), mg/g of dry weight
95
68
Deoxyribonucleic acid in lupine nodules, mg/g
0.04
0.016

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

Vitamin B12 derivatives catalyze biochemical reactions in which hydrogen transfer occurs between two adjacent carbon atoms with the simultaneous movement of other groups in the opposite direction.

Role of cobamide coenzymes in legume nodules

Application of nitrogen fixation in agriculture

Protein is an essential component of human and animal food. In many countries of the world, there is a dramatic shortage of dietary protein per unit area. Legumes provide much more protein per unit area than cereals. For example, the amount of protein in lupine and soybean seeds is 2-3 times more than in wheat, rye or oat grains, and 3-4 times more than in rice and corn grains.

The cost of legume protein is 10 times lower than that of bread cereals, while it consists of 80-90% easily digestible forms and is more complete in its composition of amino acids.

From agronomic point of view, legumes also have a great importance as crops that enrich crop residues and accumulate nitrogen in the soil, accelerate the mineralization of plant residues, increase the coefficient of utilization of soil nitrogen.

According to long-term experience conducted at the Moscow Agricultural Academy named after K.A. Timiryazev by B.A. Dospekhov, on sod-podzolic soil the introduction of clover into the crop rotation increased the rye yield by 0.75 t/ha, and additionally with the use of phosphorus and potassium fertilizers – by 1.18 t/ha. An even greater effect was achieved when lime was applied in doses of 1 t/ha on unfertilized soil. Perennial leguminous crops contribute to structure formation, prevent the development of erosion processes, and perform a phytosanitary function. For example, alfalfa suppresses cotton Verticillium wilt pathogen, so it is introduced in cotton crop rotations.

The efficiency of maximum use of biological nitrogen depends on soil acidity, i.e. the liming, application of phosphorus, potassium fertilizers and some microfertilizers.

In solving practical problems related to nitrogen nutrition, the share of biological nitrogen in the nutrient balance takes an important place. 

Data on the value 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 one third of nitrogen is fixed by legumes from the atmosphere; according to E.N. Mishustin, about 100 kg/ha of nitrogen is accumulated in alfalfa root residues per year and 50 kg/ha in clover.

According to N.S. Avdonin’s calculations, based on D.N. Pryanishnikov’s data on the ability of clover to fix 150 kg/ha of nitrogen per year, alfalfa – 250-300 kg/ha, the value of symbiotic assimilation of molecular nitrogen can reach 3 million tons, according to E.N. Mishustin – about 3.5 mln tons. On the scale of the biosphere, the role of symbiotic nitrogen fixation is small, since legumes account for about 10% of the total crop area, and in natural phytocenoses they are much smaller.

Under favorable conditions of symbiosis, i.e. with pH 6-7, providing with phosphorus, potassium, magnesium, boron, molybdenum, the presence of specific virulent strains of nodule bacteria, optimal soil moisture, pea plants fix up to 150 kg/ha, fodder beans and soybeans – up to 250 kg/ha, white lupine – up to 300 kg/ha of nitrogen, with the yield of 3-4 t/ha of seeds.

However, in practice it is rarely possible to ensure optimal conditions; symbiosis activity weakens and only 20-60 kg/ha of nitrogen is recorded, with a yield of 1.2-1.5 t/ha. Sometimes due to excessive soil acidity, lack of moisture or nutrients nitrogen fixation does not happen, the plants give low yields with minimal protein content.

There is also insufficient data on nitrogen intake into the soil with atmospheric precipitation. This value varies from 2 to 20 kg/ha per year. Up to 90% of the nitrogen that comes with seeds is used by the crop. With the seeds of legumes 8 to 15 kg/ha of nitrogen is introduced, cereals – 4-6 kg/ha.

The issues of nitrogen balance expenditure items have been studied somewhat better. Numerous works of a number of authors show that fertilizer nitrogen on various soils is used by 50-60% in the growing experiments and by 40-50% in the field. However, in determining the coefficients of nutrient use it is extremely important to study the balanced nutrition of plants with all the necessary elements.

According to V.K. Shilnikov and E.Y. Serov, the reserve on the global scale of nitrogen fixation of terrestrial and aquatic ecosystems are blue-green algae and phytosynthetic diazotrophs. Some species of cyanobacteria form associations with fungi (lichens), higher plants, such as symbiotic associations with the aquatic fern Azolla. The efficiency of many aerobic nitrogen-fixing microorganisms increases when the oxygen pressure is less than 2.02 ⋅ 104 Pa. Therefore, in seawater and groundwater, in soils of flooded rice fields, and in hot springs, favorable conditions for nitrogen fixation by aerobic microorganisms.

The phyllosphere, i.e. plant leaf surface also serves as a niche for non-symbiotic heterotrophic microorganism binding of atmospheric nitrogen. Its contribution to the total balance of nitrogen accumulation is estimated at 13-15%, and the nitrogen fixation ability of phyllospheric microorganisms at 55%.

D.N. Pryanishnikov paid much attention to the nitrogen balance as a criterion of soil fertility and crop yields. 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%, in total – 26.2%. The remaining 73.8% was a deficit.

Studying the processes of microbial nitrogen fixation will allow its more effective use in farming.

The most important practical issues of biological nitrogen that need to be studied include:

  • Ecological-biological and agronomic aspects of the process for full utilization and intensification of nitrogen fixation.
  • Study of the mechanisms of biological binding under mild conditions for the development of new methods for obtaining nitrogen fertilizers and methods for regulating nitrogen fixation processes.
  • Genetic and breeding research of legume plant symbiosis with nodule bacteria and application of genetic engineering, biochemistry and molecular biology to extend nitrogen fixation processes to many crops.

Sources

Yagodin B.A., Zhukov Y.P., Kobzarenko V.I. Agrochemistry / Edited by B.A. Yagodin. — Moscow: Kolos, 2002. — 584 p.: ill.

Plant breeding/P.P. Vavilov, V.V. Gritsenko, V.S. Kuznetsov, et al. Gritsenko, V.S. Kuznetsov, etc.; Edited by P.P. Vavilov. – M.: Agropromizdat, 1986. – 512 p.: ill. – (Textbook and textbooks for higher education institutions).