Nitrogen fertilizers are one of the highest expenses in agricultural systems and usually a limitation to the productions of many agricultural crops worldwide. The intensive use of this element in modern agriculture represents a potential environmental threat, one of the many tools for the sustainable use of this resource without losing productivity is the use of plant growth-promoting rhizobacteria, especially nitrogen-fixing bacteria. However, in considering the competitiveness of the market, studies are still needed to determine the most efficient way to use this resource and if the nitrogen mineral fertilization is indeed substitutable. As a result, this study aims to deepen the scientific knowledge of the plant-microbe interactions by addressing their main characteristics and functionalities for plant growth and development and efficiency in the use of nitrogen. For this we reviewed relevant information from scientific works that address these issues.
- nitrogen fertilizers
- nitrogen use efficiency
Nitrogen (N) is a key component of most proteins, secondary metabolites and signaling molecules . It is one of the most important macronutrients for plant development and usually one of the most limiting factor to plant production .
The use of N-fertilizers has produced a significant increase in food production in recent decades , and its consumption has grown from 11.3 Tg N year−1 in 1961 to 107.6 Tg N year−1 in 2013 . However, less than 50% of the added N is effectively absorbed by most cultivated plants [5, 6], and even N effectively converted to biomass, eventually returns to the environment . In the soil, N is available to plants in the form of nitrate (NO3−), ammonium (NH4+) and organic compounds (usually amino acids), being the NO3, the most abundant . In its ionic form, NO3− has a negative charge and high water solubility, being susceptible to leaching and runoff . It can also be volatilized by denitrifying microorganisms , and lost to the atmosphere in the form of nitrous oxide (N2O, a greenhouse gas 296-fold more potent than a unit of CO2). Leaching of N causes eutrophication of water bodies and contamination of groundwater .
N can also be lost to the atmosphere in other forms such as reactive gases (NOx; NH3), aggravating the greenhouse effect , and is also related to the acidification of soils through the formation of acid rain, and depletion of exchangeable basic soil cations .
To minimize the loss of N in the soil, and consequently the total amount of N necessary for a high-quality production, several strategies have been used. One is the use of urease inhibitors such as N-(n-butyl)-thiophosphoric triamide (NBPT) to delay the hydrolysis of urea, thus reducing losses to the atmosphere and microbial transformations . Increased N use efficiency (NUE) has also been the subject of research, through the selection of genotypes with a higher NUE  or through biological nitrogen fixation . Biological nitrogen fixation (BNF) occurs through the conversion of atmospheric N2 to ammonium by free-living or symbiotic diazotrophic bacteria . Plant growth-promoting rhizobacteria (PGPR) can increase the N absorption capacity through BNF , phytohormone production , stimulate the production and enzymatic assimilation of NH4+ , as well as the transport and partition of N .
The study of plant nutrition related to the use of BNF as an alternative to increase efficiency and sustainability in the use of N in agriculture is essential, given the complex nature of the interactions between soil, plant, and microorganisms. Therefore, the objective of this review is to deepen the scientific knowledge of these interactions, addressing their main characteristics and functionalities for plant growth and development.
2. Mechanisms of biological nitrogen fixation
One of the largest N reservoirs is the atmosphere, second only to the lithosphere in absolute amount of N . N makes up about 78% of the atmosphere  and is mainly in the form of molecular N (N2). The atoms in the N2 molecule have low-energy orbitals and the bond between the two N molecules is relatively short (1,098 Å) and stable, with a bonding energy of 930 kJ/mol . This set of characteristics gives low reaction potential to the molecule. Alternatively, N2 can be reduced to NH3 naturally by microorganisms through the BNF process. The BNF reaction follows the following stoichiometry:
The BNF process is catalyzed exclusively by an enzyme complex called the nitrogenase complex. The nitrogenase complex is composed of dinitrogenase reductase (Iron-protein) and dinitrogenase (Molybdenum-Iron-protein) (Figure 1a). Dinitrogenase-reductase is a dimer of approximately 60 kDa, composed of two identical and symmetrical subunits, which coordinate a redox center 4Fe-4S (Figure 2). This enzyme also has sites for the binding of ATP/ADP, one in each subunit, being able to couple the hydrolysis of ATP to fuel the transfer of electrons to the dinitrogenase . Dinitrogenase is a heterotetramer α2β2 with approximately 240 kDa.
Dinitrogenase has two cofactors containing iron (Figure 1b), being the group P and the FeMo cofactor . Group P contains a pair of 4Fe-4S centers, which share a sulfur, forming an 8Fe-7S center. The FeMo cofactor is a variation of the iron–sulfur groups, such as the P group, but differs greatly from the other metallic sites within this family. Its structure is composed of [Mo: 7Fe: 9S: C]: Homocitrate [26, 28]. Some microorganisms also have alternative forms of the MoFe cofactor, where Molybdenum (Mo) is replaced by atoms of Vanadium (V) or Iron (Fe) depending on metal availability .
In the N2 reduction reaction (Eq. 1), the reduced dinitrogenase reductase couples the hydrolysis of ATP with the transfer of electrons to the dinitrogenase. The oxidized dinitrogenase reductase detaches from the dinitrogenase, only to be reduced again (by ferredoxins or flavodoxins). Again, there is the coupling between both enzymes followed by the consumption of ATP to transfer the electrons to the dinitrogenase. This coupling-detaching cycle followed by the electron transfer is repeated until the dinitrogenase is reduced enough to reduce its substrate, which in the case of BNF, is N2 .
Because it deals with large amounts of energy, enough to break the triple bond of the N2 molecule, the nitrogenase complex is not only inactivated by the presence of oxygen (O2) but can have its expression reduced . However, diazotrophic bacteria are able to combine N fixation with their aerobic metabolism in different ways to avoid O2 deactivation . Notably, one of the most advanced means of controlling O2 concentration is expressed by rhizobia-leguminous symbiosis.
3. Brazil: a success of BNF in legumes
Brazil is one of the best examples in the efficient research and use of the BNF in legumes . The use of FBN is interesting from both an economic and an environmental point of view, since once this process is established, nitrogen fertilizers can be dispensed with in whole or in part, thus contributing to enable reforestation and minimize possible environmental impacts resulting of use these supplies . It is estimated an annual economy of more than US$ 13 billion with the total or partial substitution of nitrogen fertilizers in legumes in Brazil .
Rhizobium-leguminous symbiosis is the most important symbiotic system between microorganisms and plants thanks to the efficiency of the N2 fixation process, and one of the justifications is in the amplitude and geographical distribution of the hosts and the economic impact it causes in agriculture, and one of the main sources of N for the biosphere . The leguminosae family comprises almost 20 thousand species, including tree, herbaceous species used as fodder, producers of raw materials or directly in human food .
The most successful case here in Brazil according to Hungria et al. , is the symbiosis of Bradyrhizobium spp. with soybean (
Alfalfa is another plant species with a high potential for biological nitrogen fixation. As a legume, alfalfa is capable of symbiotically associating with rhizobial bacteria, with N inputs to reaching up to 470 kg of N ha-1 . The main symbiotic alfalfa bacteria belong to the genus Ensifer, having Synorhizobium as a synonym, but previously classified as Rhizobium. Sinorhizobium meliloti and Sinorhizobium mediace are the main symbionts reported in several countries . In Brazil, there are three strains of rhizobia that are used in commercial inoculants for alfalfa, which have been validated for more than two decades and with rare tests conducted with the same .
4. Growth promotion by associative and free-living diazotrophic bacteria and stimulating N metabolism
World production is dominated by the production of four grasses (FAO Stats 2019), namely: Sugarcane (
Grasses are of great interest for the development of biological nitrogen fixation aiming at greater efficiency, in view of its relatively low NUE [43, 44]. The increase in NUE is related to several characteristics, Iqbal et al., (2020) working with different cotton genotypes analyzed the biochemical and morphological responses of the accessions according to different concentrations of NO3− and the root architecture and efficiency of the enzymes of the assimilation of N were essential for the increase in traction related to NUE. The inoculation of diazotrophic bacteria seems to achieve the same results in different cultures of importance to the global market such as sugarcane (
Modulation of root architecture induced by PGPR is also a morphological trait essential to the increase in NUE. The increase in the area explored by the root triggered by the increase in the volume of the roots caused by these bacteria directly influences the interception of nutrients, among them the N. Besides the influence on the morphological characteristics, the PGPR inoculation has an impact on the metabolism activity of the N. The increase in these characteristics makes these bacteria a potential solution for increasing NUE.
Since the discovery of the Haber-bosch process in the early 20th century, the levels of N added to the biosphere continue to increase each year and its excessive use of this element is a source of numerous environmental problems.
Therefore, biological nitrogen fixation process is an essential tool in the current economic, agricultural, and environmental context of many countries, this can be seen through studies and government data that show a reduction in financial expenses in the order of millions, this technological tool. it is a reality in vegetable crops with high potentials in the agricultural network of emerging powers worldwide, in addition to contributing to the reduction of potentially harmful agents for the worsening of the greenhouse effect.
This technology has been extensively scientifically explored, aiming to expand its possibility in other promising cultures in the agricultural and forestry world, as well as other associative and free-living diazotrophic microorganisms, and their ability to promote plant growth. However, there are still major gaps in knowledge about the diversity and mechanisms of PGPR action and further research is needed to establish the use of these new bacteria as a sustainable agricultural practice.
To Laboratory of Biodiversity Studies of Upper Plants in Federal Rural University of Amazonia.
Appendices and nomenclature
National Supply Company Nitrogen use efficiency N-(n-butyl)-thiophosphoric triamide Plant growth-promoting rhizobacteria Biologic nitrogen fixation
National Supply Company
Nitrogen use efficiency
Plant growth-promoting rhizobacteria
Biologic nitrogen fixation