Biological Nitrogen Fixation Is Carried Out By

Biological nitrogen fixation, a crucial process for life on Earth, is the conversion of atmospheric nitrogen (N₂) into ammonia (NH₃), a form of nitrogen that plants and other organisms can utilize. This remarkable feat is primarily carried out by a select group of microorganisms, showcasing the intricate interplay between biology and chemistry in sustaining ecosystems.

The Significance of Biological Nitrogen Fixation

Nitrogen is an essential element for all living organisms, serving as a key component of proteins, nucleic acids (DNA and RNA), and other vital biomolecules. While nitrogen is abundant in the atmosphere, existing as approximately 78% of the air we breathe, plants cannot directly use it in its gaseous form. They require nitrogen to be in a "fixed" form, such as ammonia, nitrate (NO₃⁻), or organic nitrogen compounds.

Biological nitrogen fixation (BNF) is a critical natural process that bridges this gap, converting inert atmospheric nitrogen into biologically available forms. This process is particularly important in agriculture and natural ecosystems, where it can be a limiting factor for plant growth and overall productivity. Without BNF, many ecosystems would struggle to sustain life, and agricultural yields would be significantly lower.

The Microorganisms Responsible for Nitrogen Fixation

The microorganisms responsible for biological nitrogen fixation are called diazotrophs. These organisms possess a unique enzyme complex called nitrogenase, which catalyzes the reduction of atmospheric nitrogen to ammonia. Diazotrophs are a diverse group of bacteria and archaea, found in various habitats, including soil, water, and plant tissues.

Here's a closer look at the major groups of diazotrophs:

• Free-Living Diazotrophs: These bacteria live independently in the soil or water and fix nitrogen without forming a direct symbiotic relationship with plants. Examples include:
  • Azotobacter: Aerobic bacteria commonly found in soil.
  • Azospirillum: Microaerophilic bacteria that can associate with plant roots.
  • Clostridium: Anaerobic bacteria found in soil and sediments.
  • Cyanobacteria (blue-green algae): Photosynthetic bacteria found in aquatic and terrestrial environments.
• Symbiotic Diazotrophs: These bacteria form mutually beneficial relationships with plants, where the bacteria fix nitrogen for the plant in exchange for carbon and other nutrients. The most well-known example is:
  • Rhizobium: Bacteria that form nodules on the roots of leguminous plants (e.g., beans, peas, soybeans).
  • Frankia: Bacteria that form nodules on the roots of actinorhizal plants (e.g., alder, casuarina).
• Associative Diazotrophs: These bacteria live in close association with plants, either on the root surface (epiphytically) or within the root tissues (endophytically), and fix nitrogen for the plant. Examples include:
  • Azotobacter and Azospirillum: As mentioned above, these can also function as associative diazotrophs.
  • Gluconacetobacter diazotrophicus: An endophytic bacterium found in sugarcane and other plants.

The Nitrogenase Enzyme Complex

The nitrogenase enzyme complex is the key to biological nitrogen fixation. It is a highly conserved enzyme found in all diazotrophs, although there can be variations in its structure and metal composition. The nitrogenase complex consists of two main protein components:

• Dinitrogenase Reductase (Fe Protein): This smaller protein contains iron-sulfur clusters and is responsible for transferring electrons to the dinitrogenase component. It is highly sensitive to oxygen and is often protected by specialized cellular structures or metabolic strategies.
• Dinitrogenase (MoFe Protein): This larger protein contains a molybdenum-iron cofactor (FeMo-co) at its active site, where nitrogen reduction occurs. The FeMo-co is a complex cluster of iron, molybdenum, sulfur, and carbon atoms, and it is unique to the nitrogenase enzyme.

The nitrogenase enzyme catalyzes the following reaction:

N₂ + 8H⁺ + 8e⁻ + 16ATP → 2NH₃ + H₂ + 16ADP + 16Pi

This equation shows that the reduction of one molecule of nitrogen gas (N₂) to two molecules of ammonia (NH₃) requires eight protons (H⁺), eight electrons (e⁻), and a significant amount of energy in the form of adenosine triphosphate (ATP). The reaction also produces one molecule of hydrogen gas (H₂).

The high energy requirement for nitrogen fixation is due to the strong triple bond between the two nitrogen atoms in N₂. Breaking this bond and reducing the nitrogen atoms to ammonia requires a significant input of energy.

The Process of Biological Nitrogen Fixation

The process of biological nitrogen fixation can be broken down into several key steps:

1. Nitrogenase Synthesis: Diazotrophs must first synthesize the nitrogenase enzyme complex. This process is tightly regulated and is typically only activated when fixed nitrogen is scarce in the environment.
2. Electron Transfer: Electrons are transferred from a reducing agent (e.g., ferredoxin or flavodoxin) to the dinitrogenase reductase (Fe protein). This protein then transfers the electrons to the dinitrogenase (MoFe protein).
3. Nitrogen Reduction: The dinitrogenase enzyme binds atmospheric nitrogen (N₂) at its active site (FeMo-co). Through a series of electron transfers and proton additions, the nitrogen molecule is gradually reduced to ammonia (NH₃).
4. Ammonia Assimilation: The ammonia produced by nitrogen fixation is then assimilated into organic compounds, such as glutamate and glutamine, which are used to synthesize other nitrogen-containing biomolecules.

Factors Affecting Biological Nitrogen Fixation

Several factors can influence the rate and efficiency of biological nitrogen fixation:

• Oxygen: Nitrogenase is extremely sensitive to oxygen, as oxygen can irreversibly damage the enzyme. Diazotrophs have evolved various mechanisms to protect nitrogenase from oxygen, including:
  • Respiration: Rapidly consuming oxygen to create a low-oxygen environment.
  • Heterocyst Formation: Specialized cells in cyanobacteria that lack oxygen-evolving photosynthesis.
  • Leghemoglobin: An oxygen-binding protein in legume nodules that regulates oxygen levels.
• Molybdenum and Iron: Molybdenum and iron are essential components of the nitrogenase enzyme. Their availability in the environment can limit nitrogen fixation.
• pH: Nitrogen fixation is generally optimal at a neutral pH. Acidic or alkaline conditions can inhibit nitrogenase activity.
• Temperature: Nitrogen fixation is generally more efficient at warmer temperatures, up to a certain point. Extreme temperatures can denature the nitrogenase enzyme.
• Nutrient Availability: The availability of other nutrients, such as phosphorus, potassium, and micronutrients, can also affect nitrogen fixation.
• Fixed Nitrogen: The presence of fixed nitrogen in the environment can inhibit nitrogen fixation, as diazotrophs will preferentially use available fixed nitrogen rather than fixing it themselves.
• Water Availability: Water stress can reduce nitrogen fixation rates by affecting plant growth and the activity of diazotrophs in the soil.

Symbiotic Nitrogen Fixation: The Rhizobium-Legume Symbiosis

The symbiotic relationship between Rhizobium bacteria and leguminous plants is one of the most important examples of biological nitrogen fixation. This symbiosis allows legumes to thrive in nitrogen-poor soils and is crucial for sustainable agriculture.

The process of nodule formation and symbiotic nitrogen fixation involves a complex series of interactions between the plant and the bacteria:

1. Recognition and Attachment: The plant roots release signaling molecules (flavonoids) that attract Rhizobium bacteria. The bacteria attach to the root hairs and initiate the infection process.
2. Nodule Formation: The bacteria enter the root hairs and travel through the plant cells to the root cortex, where they stimulate the formation of a nodule. The nodule is a specialized structure that provides a protected environment for the bacteria to fix nitrogen.
3. Bacteroid Differentiation: Inside the nodule, the Rhizobium bacteria differentiate into bacteroids, which are specialized nitrogen-fixing cells.
4. Nitrogen Fixation: The bacteroids fix nitrogen for the plant, converting atmospheric nitrogen into ammonia. The ammonia is then assimilated into amino acids and other nitrogen-containing compounds, which are transported to the rest of the plant.
5. Nutrient Exchange: In exchange for nitrogen, the plant provides the bacteroids with carbon and other nutrients, such as sugars and organic acids.

The Rhizobium-legume symbiosis is highly specific, with different species of Rhizobium forming nodules with different species of legumes. This specificity is determined by the exchange of signaling molecules between the plant and the bacteria.

Applications of Biological Nitrogen Fixation

Biological nitrogen fixation has numerous applications in agriculture and environmental management:

• Biofertilizers: Diazotrophs can be used as biofertilizers to enhance crop yields and reduce the need for synthetic nitrogen fertilizers. Biofertilizers can be applied to the soil or used to inoculate seeds.
• Crop Rotation: Rotating crops with legumes can improve soil fertility by adding fixed nitrogen to the soil. This practice is commonly used in sustainable agriculture.
• Cover Cropping: Planting cover crops, such as legumes, can help to prevent soil erosion, suppress weeds, and improve soil fertility through nitrogen fixation.
• Reforestation: Planting actinorhizal plants, such as alder and casuarina, can improve soil fertility in degraded lands and promote reforestation.
• Wastewater Treatment: Some diazotrophs can be used to remove nitrogen from wastewater, helping to prevent eutrophication of aquatic ecosystems.

The Future of Biological Nitrogen Fixation Research

Research on biological nitrogen fixation is ongoing, with the goal of improving the efficiency and expanding the application of this important process. Some key areas of research include:

• Understanding the Molecular Mechanisms of Nitrogen Fixation: Researchers are working to understand the detailed molecular mechanisms of nitrogenase and how it is regulated. This knowledge could be used to engineer more efficient nitrogen-fixing enzymes.
• Improving the Efficiency of Symbiotic Nitrogen Fixation: Researchers are trying to improve the efficiency of the Rhizobium-legume symbiosis by selecting for more effective strains of Rhizobium and developing legume varieties that are more compatible with these strains.
• Expanding the Range of Nitrogen-Fixing Plants: Researchers are exploring the possibility of transferring nitrogen-fixing genes from bacteria to non-leguminous crops, such as cereals. This would reduce the need for synthetic nitrogen fertilizers in these crops.
• Developing New Biofertilizers: Researchers are developing new biofertilizers that contain a diverse range of diazotrophs and other beneficial microorganisms. These biofertilizers could be more effective than traditional biofertilizers.
• Understanding the Role of Nitrogen Fixation in Natural Ecosystems: Researchers are studying the role of nitrogen fixation in various natural ecosystems, such as forests, grasslands, and wetlands. This knowledge is important for understanding how these ecosystems function and how they are affected by environmental changes.

Conclusion

Biological nitrogen fixation is a vital process carried out by a diverse group of microorganisms called diazotrophs. These organisms convert atmospheric nitrogen into ammonia, a form of nitrogen that plants and other organisms can utilize. Nitrogen fixation is essential for maintaining soil fertility, supporting plant growth, and sustaining ecosystems.

The nitrogenase enzyme complex is the key to biological nitrogen fixation. This enzyme catalyzes the reduction of atmospheric nitrogen to ammonia, a process that requires a significant input of energy. Diazotrophs have evolved various mechanisms to protect nitrogenase from oxygen, which is a potent inhibitor of the enzyme.

The symbiotic relationship between Rhizobium bacteria and leguminous plants is one of the most important examples of biological nitrogen fixation. This symbiosis allows legumes to thrive in nitrogen-poor soils and is crucial for sustainable agriculture.

Biological nitrogen fixation has numerous applications in agriculture and environmental management, including the use of biofertilizers, crop rotation, cover cropping, reforestation, and wastewater treatment. Research on biological nitrogen fixation is ongoing, with the goal of improving the efficiency and expanding the application of this important process. As we face increasing challenges related to food security and environmental sustainability, understanding and harnessing the power of biological nitrogen fixation will be crucial for creating a more sustainable future.