Nitrogen Fixation

Nitrogen fixation is the process by which molecular nitrogen gas (<span class="katex"><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:0.8333em;vertical-align:-0.15em;"></span><span class="mord"><span class="mord mathnormal" style="margin-right:0.10903em;">N</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.3011em;"><span style="top:-2.55em;margin-left:-0.109em;margin-right:0.05em;"><span class="pstrut" style="height:2.7em;"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight">2</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.15em;"><span></span></span></span></span></span></span></span></span></span>), which is abundant in the atmosphere but unusable by most living organisms, is converted into forms such as ammonium (<span class="katex"><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.0778em;vertical-align:-0.2663em;"></span><span class="mord mathnormal" style="margin-right:0.10903em;">N</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.08125em;">H</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.8115em;"><span style="top:-2.4337em;margin-left:-0.0813em;margin-right:0.05em;"><span class="pstrut" style="height:2.7em;"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight">4</span></span></span></span><span style="top:-3.1031em;margin-right:0.05em;"><span class="pstrut" style="height:2.7em;"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight">+</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2663em;"><span></span></span></span></span></span></span></span></span></span>) that plants and other organisms can utilize. Nitrogen is an essential nutrient for all living organisms because it is a structural component of fundamental organic compounds such as proteins, nucleic acids, chlorophyll, and enzymes. Despite constituting approximately 78% of the atmosphere, nitrogen deficiency is one of the most common nutrient limitations. Nitrogen fixation is the primary mechanism enabling atmospheric nitrogen to enter the biosphere. This process can occur through non-biological (atmospheric), industrial, and biological pathways.

Methods of Nitrogen Fixation

Non-Biological Nitrogen Fixation

This process, also known as atmospheric nitrogen fixation, occurs during high-energy natural atmospheric events such as lightning and thunderstorms. These events break the strong triple bond between nitrogen molecules in the atmosphere, allowing nitrogen to combine with oxygen to form nitrogen oxides. These compounds are subsequently deposited into the soil via rainfall as nitrate (<span class="katex"><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.0778em;vertical-align:-0.2663em;"></span><span class="mord mathnormal" style="margin-right:0.10903em;">N</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.02778em;">O</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.8115em;"><span style="top:-2.4337em;margin-left:-0.0278em;margin-right:0.05em;"><span class="pstrut" style="height:2.7em;"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight">3</span></span></span></span><span style="top:-3.1031em;margin-right:0.05em;"><span class="pstrut" style="height:2.7em;"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight">−</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2663em;"><span></span></span></span></span></span></span></span></span></span>) and ammonium (<span class="katex"><span class="katex-html" aria-hidden="true"><span class="base"><span class="strut" style="height:1.0778em;vertical-align:-0.2663em;"></span><span class="mord mathnormal" style="margin-right:0.10903em;">N</span><span class="mord"><span class="mord mathnormal" style="margin-right:0.08125em;">H</span><span class="msupsub"><span class="vlist-t vlist-t2"><span class="vlist-r"><span class="vlist" style="height:0.8115em;"><span style="top:-2.4337em;margin-left:-0.0813em;margin-right:0.05em;"><span class="pstrut" style="height:2.7em;"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight">4</span></span></span></span><span style="top:-3.1031em;margin-right:0.05em;"><span class="pstrut" style="height:2.7em;"></span><span class="sizing reset-size6 size3 mtight"><span class="mord mtight"><span class="mord mtight">+</span></span></span></span></span><span class="vlist-s">​</span></span><span class="vlist-r"><span class="vlist" style="height:0.2663em;"><span></span></span></span></span></span></span></span></span></span>). The amount of nitrogen fixed by this method is lower compared to other fixation types. It is estimated that the amount of nitrogen added to the soil via precipitation ranges from 1.8 to 38 kilograms per hectare annually.

Industrial Nitrogen Fixation

Industrial nitrogen fixation is primarily achieved through the Haber-Bosch process, which produces synthetic ammonia-based fertilizers. This process requires high temperatures (400°C) and high pressures (200–350 atm). This substantial energy demand is largely met by non-renewable fossil fuels such as petroleum. Despite its high energy input, approximately 40 million tons of nitrogen are produced annually through this method.

Biological Nitrogen Fixation (BNF)

Biological nitrogen fixation is the conversion of atmospheric molecular nitrogen into ammonia by certain microorganisms possessing the enzyme nitrogenase. This process accounts for approximately 75% of total nitrogen fixation globally, with an estimated 175 million tons of nitrogen fixed annually through this route. Biological nitrogen fixation is considered one of the most important biochemical processes on Earth after photosynthesis. It occurs primarily through two main mechanisms: symbiotic and non-symbiotic.

Non-Symbiotic (Asymbiotic) Fixation

This type of fixation is carried out by free-living microorganisms in soil and aquatic ecosystems. It is estimated that approximately 30 million tons of nitrogen are fixed annually through this method. The main groups of microorganisms performing non-symbiotic fixation include:

• Heterotrophic bacteria: Genera such as Azotobacter, Clostridium, Bacillus, and Pseudomonas.

It has been reported that Azotobacter species can fix up to 90 kilograms of nitrogen per hectare annually under optimal conditions.

• Cyanobacteria (blue-green algae): Species such as Anabaena, Nostoc, and Calothrix, which are particularly effective in rice paddies, belong to this group. These organisms are reported to fix 100–300 kilograms of nitrogen per hectare annually.

• Photosynthetic bacteria: Includes species such as Chlorobium and Rhodomicrobium.

• Chemoautotrophic bacteria: An example is the genus Methanobacillus.

Symbiotic Fixation

Symbiotic nitrogen fixation occurs through a mutually beneficial relationship between a microorganism and a host plant. This process accounts for approximately half of all biologically fixed nitrogen. The best-known symbiotic relationship is established between bacteria of the genus Rhizobium and plants of the family Leguminosae. In this relationship, the bacterium receives carbohydrates from the host plant as an energy source and, in return, supplies the plant with nitrogen fixed from the atmosphere. The bacteria form specialized structures called "nodules" on the plant roots, where nitrogen fixation takes place.

This symbiosis is highly specific; each Rhizobium species interacts with a particular group of legumes. Examples include:

• Rhizobium leguminosarum: Pea, vetch, lentil.
• Rhizobium trifolii: Clover.
• Rhizobium meliloti: Alfalfa.
• Bradyrhizobium japonicum: Soybean.
• Rhizobium cicer: Chickpea.

Through the legume-Rhizobium symbiosis, between 200 and 300 kilograms of nitrogen per hectare annually can be made available to the plant, with some cases reaching up to 600 kilograms. Alfalfa is known as one of the plants with the highest nitrogen fixation capacity, capable of fixing 125–335 kilograms of nitrogen per hectare annually.

Nodule Formation Process (Nodulation)

The formation of nodules where symbiotic fixation occurs proceeds through three main stages:

1. Pre-infection Phase: Plant roots secrete chemical compounds that attract bacteria. Bacteria adhering to root hairs secrete hormones such as indole acetic acid (IAA), inducing specialized root hair deformation and producing the enzyme polygalacturonase (PG) to soften the root cell wall for bacterial entry.
2. Formation of Infection Thread: Bacteria entering root cells form a tubular structure called an "infection thread" or "filament," which extends toward the cortex. Bacteria multiply within this thread and are transported into the cortex.
3. Nodule Formation: Upon reaching cortical cells, the infection thread triggers rapid cell division in these and neighboring cells. Bacteria lose their typical rod shape inside the host cells and transform into irregular, nitrogen-fixing forms called "bacteroids."

Nitrogen fixation begins approximately 10–21 days after infection. An active, nitrogen-fixing nodule appears reddish or pinkish when cut open due to the presence of the pigment leghemoglobin. A greenish or brownish internal color indicates that the nodule is inactive.

Bacterial Inoculation

When a particular legume crop is being grown for the first time in a field, the soil may not contain sufficient numbers of effective Rhizobium bacteria specific to that plant. In such cases, "bacterial inoculation" is required to ensure effective nitrogen fixation. Inoculation involves introducing a bacterial culture with high nitrogen-fixing capacity to the seedbed or directly to the seeds.

Soil Inoculation

This method involves spreading soil collected from a field previously cultivated with the same legume onto the new field. However, this approach is less preferred because it requires large quantities of soil (up to 400 kg per decare), is costly, and carries the risk of introducing pathogens, pests, and weed seeds along with the soil.

Seed Inoculation

This is the most common and practical method. In this approach, a bacterial culture (usually in a peat-based carrier) is applied to slightly moistened seeds. A sticky agent such as sugar water may be used to improve bacterial adhesion to the seed. Inoculated seeds must be protected from direct sunlight and planted within a few hours at the latest to prevent loss of bacterial viability.

Research has shown that alternative methods such as seedbed pulverization inoculation can be more effective than traditional seed inoculation. Inoculated seeds must not be directly exposed to lime or inorganic fertilizers.

Factors Affecting Nitrogen Fixation

The efficiency of biological nitrogen fixation is influenced by a range of genetic and environmental factors.

Bacterial Strain and Host Compatibility

A genetic compatibility between the host plant and the Rhizobium strain is essential for fixation to occur. The fixation efficiency of different bacterial strains can vary on the same plant species.

Nutrient Elements

• Nitrogen (N): High levels of mineral nitrogen (particularly nitrate) in the soil inhibit nodule formation and the activity of nitrogenase. Low initial nitrogen levels, however, can promote nodulation by supporting early plant development.

• Phosphorus (P) and Potassium (K): Phosphorus supports early nodule formation by enhancing root development and bacterial activity. Potassium also has positive effects on nodulation and nitrogen fixation.

Temperature

Both low and high temperatures negatively affect fixation. Low temperatures delay root infection and inhibit nitrogenase activity. Generally, temperatures above 30°C reduce nodule formation and activity.

Soil Moisture (Drought and Waterlogging)

Symbiotic nitrogen fixation is highly sensitive to drought. Water stress reduces nodule number and size, limits the transport of photosynthetic products to nodules, and decreases nitrogenase activity. Excess soil water reduces oxygen levels in the root zone, impairing nodule formation and function.

Soil pH

Rhizobium bacteria generally perform best under near-neutral conditions (pH 6.8). Low pH (acidic soils) inhibits bacterial growth, hinders root infection, and can lead to toxic levels of elements such as aluminum (Al) and manganese (Mn).

Salinity

High salt concentrations in the soil cause osmotic stress (physiological drought) and ion toxicity, negatively affecting both the host plant and the bacteria. Salinity reduces root hair infection, nodule number, nodule respiration, and leghemoglobin content.

Atmospheric Carbon Dioxide (CO₂) Increase

Increasing atmospheric CO₂ concentrations enhance photosynthesis in plants. This leads to greater transport of carbohydrates (energy) to roots and nodules, promoting nodule development, increasing nodule number and size, and stimulating nitrogenase activity. Consequently, elevated CO₂ conditions generally have a positive effect on biological nitrogen fixation.