How to increase biological nitrogen fixation in soybeans?

Learn more about optimizing soybean growth with tips for evaluating biological nitrogen fixation symbiosis and nodulation.

April 16, 2024
6 mins
Eduardo Lopes Cancellier
Biostimulants Agronomy Specialist, ICL

Biological nitrogen fixation (BNF) in soybean crops occurs through a symbiosis between bacteria of the genus Bradyrhizobium and soybean plants. This process between the plant and the bacteria can be observed by the formation of nodules on the roots in the initial phase and obtaining full establishment from the V4 phenological stage.

At this point, it is very important to evaluate nodulation performance: the soybean root must contain at least 10 to 15 active nodules and measure 3 to 8 mm with a pink color, indicating that the bacteria is alive and breathing (Câmara, 2014).

For BNF to occur efficiently, the soil must have balanced fertility to ensure greater availability of nutrients essential to the process. Therefore, each nutrient has specific functions directly or indirectly linked to FBN, as described in Table 1.

Table 1. Mineral nutrients, functions and consequences of deficiencies for BNF.

NutrientFunctions at FBNEffects of element deficiency on nodulation
BoronCell division.Decrease in nodule sizes.
PhosphorusDirectly related to the production and consumption of ATP.Reduction of nodulation and BNF, inducing N deficiency.
CalciumRoot growth and action on chemical signal messenger proteins.Reduction of nodulation due to reduction in root surface.
SulfurConstituent of secondary metabolites that act on soybean nodulation factors (Nod).Delay and reduction in root nodulation.
NickelConstituent of plant enzymes (urease) and required for H2 reprocessing (hydrogenase).Delay in nodulation and reduction in BNF.
MolybdenumConstituent of molybdate-iron-protein nitrogenase.Ineffective nodulation and N deficiency.
IronConstituent of nitrogenase iron-protein.Delayed onset, decreased nodulation and reduced BNF.
MagnesiumA constituent of chlorophyll, it has a direct relationship.N deficiency induced by reduction of BNF.
CobaltComponent of cobalamin (B12), precursor of leghemoglobin.Delayed onset and decrease in nodulation.
ZincIt acts in the synthesis of Leghemoglobin.Reduction in the number and size of nodules.
CopperNot yet understood.Reduction in BNF.
PotassiumActivator of several enzymes in photosynthesis and respiration.Reduction in the dry mass of nodules with reduction in BNF.

Did you know?

It is possible to obtain high soybean productivity without top dressing and/or foliar nitrogen application. For each bag of soybean produced, the plant needs approximately 4.9 kg of N, that is, if a productivity of 80 sc/ha is considered, it can accumulate up to 392 kg/ha of N. This will be fully supplied via BNF and mineralization of soil N. This process is possible as long as it offers nutrients and favorable conditions for the full development of the bacteria in the nodules (Table 1).

Figure 1. Variation in nodulation during the soybean phenological cycle, expressed in total number of nodules on roots of 10 plants. Source: Câmara, 2014.

Throughout the soybean cycle, root nodules are constantly formed and renewed, making the phenomenon extremely dynamic and responsive to physiological effects. Thus, nutritional and physiological stimuli, in addition to inoculation, carried out in seed treatment (ST), are responsible for the formation of nodules that will supply the first peak of N demand in soybeans, at the R1 phenological stage (beginning of flowering).

The second nutritional and physiological stimulus must occur in V4, in the search for the renewal of nodules that will meet the N demand in the R5.3 stage (grain filling) (Figure 1).

To calculate the BNF, we use advanced techniques with 15N isotopes with the aim of directly calculating the % of BNF in the soybean crop.

To achieve this, the natural abundance technique of 15N is used. This technique is premised on the difference between the 15N present in atmospheric N and soil N, with soil N being slightly enriched. Thus, the resulting 15N abundance in soybean N is the ratio between soil N sources (mineralization) and atmospheric N (via BNF). Lavres et al., 2016 show more details of this technique.


The 2019 CESB (Brazilian Soy Strategic Committee) national champion, producer Maurício De Bortolli | Aurora Seeds, Cruz Alta-RS, produced 123.8 sc/ha of soybeans without applying N. He used ICL technologies included in the crop application program, that stimulates BNF process, balanced nutrition, stress reduction, greater fruit set flowers and grain filling.

Figure 2. Percentage of biological N fixation (%BNF) estimated using the natural abundance technique of 15N in soybeans evaluated in R5 under field conditions.

When evaluating the direct impact of our portfolio on BNF in soybeans, a study was carried out using the technique previously described. This study included the following treatments: Control, Bioz Topaz, Nova PULSE (sold outside of the US as BIOZ Nephrite) and BIOZ Topaz + Nova PULSE. Inoculant and fungicides were applied to all treatments, with the variation factor being the presence or absence of Topaz and Nova PULSE.

Thus, it was possible to demonstrate that the application of BIOZ Topaz + Nova PULSE, provided a 33% increase in BNF compared to the control treatment (Figure 2). In general, nodules have a longevity that can vary from 6 to 8 weeks. As the crop’s nitrogen demand curve lasts longer than the lifespan of the nodules, it is essential not only to stimulate the formation of nodules, but also their longevity and renewal. With this in mind, Nova  PULSE was developed with the function of assisting in the renewal of nodules and promoting nodule activity at the time of greatest demand for the R5 plant (Figure 3). With the application of Up! Seeds® + Tônus® showed a 20% increase in the number of nodules in relation to the control treatment, which highlighted its influence on the renewal of nodules (Figure 3).

Figure 3. Numbers of nodules per plant in the V6 and R5 stages of soybeans compared with products for biostimulation in the seeds (Topaz) and physiological action(Nephrite) applied in V4, experiments carried out under field conditions (ICL Innovation Center).

In this way, the benefits of BIOZ Topaz (150 mL/ha) in seed treatment, stimulating rooting and BNF in soybeans, increasing the number of nodules (Figure 4) and contributing to plant development. To stimulate nodules renewal and to meet N demand in reproductive stages, the importance Nova PULSE application (300 g/ha) in the V4 phase was highlighted.

This product enhances the activity of nitrogenase and hydrogenase enzymes, which are key in the FBN process. It also stimulates nitrate reductase and urease, essential enzymes for N metabolism (Lavres et al., 2016), in addition to containing compounds with physiological effects that are auxin precursors that stimulate the growth of the root system, facilitating the formation and renewal of nodules.

Figure 4. Mass (g) of nodules obtained from an experiment with the above treatments (data from 5 plants per treatment). Photo: GIL CÂMARA (2019).

This product enhances the activity of nitrogenase and hydrogenase enzymes, which are key in the BNF process. It also stimulates nitrate reductase and urease, essential enzymes for N metabolism (Lavres et al, 2016). In addition, it contains compounds with physiological effects working as auxin precursors that stimulate the growth of the root system, facilitating the formation and renewal of nodules.


José Marcos Leite – Research Manager

Robson Mauri – Technical and Innovation Manager

Rafael Butke Baptista – Product Manager

Bibliographic references:

CAMARA, GMS Biological nitrogen fixation in soybeans. Agronomic information, n. 7, 2014.

HUNGARY, M.; CAMPO, RJ; MENDES, IC The importance of the biological nitrogen fixation process for soybean cultivation: essential components for the competitiveness of the Brazilian product. Londrina: Embrapa Soja, 2007. 80p. (Embrapa Soja. Documents, 283).

LAVRES, JOSÉ; CASTRO FRANCO, GUILHERME ; DE SOUSA CÂMARA, GIL M. . Soybean Seed Treatment with Nickel Improves Biological Nitrogen Fixation and Urease Activity. Frontiers in Environmental Science, vol. 4, p. 1-11, 2016