Biological Nitrogen Fixation

Biological Nitrogen Fixation

Overview

• Biological Nitrogen Fixation (BNF) is a crucial process within the nitrogen cycle.
• The aerial part of the plant is referred to as the phyllosphere.
• The area surrounding the roots is referred to as the rhizosphere.
• The endosphere refers to the inside of the plant.
• 78% of the atmosphere consists of N2 gas.
• N2 is reduced to NH3 (ammonia) through biological or industrial N2 fixation.
• Bacteria play a key role in BNF.
• Nitrogen-containing fertilizers are used for plants.
• Excess NH3 is processed by microorganisms through:
  • Nitrification
  • Denitrification
• These processes can sometimes cause environmental pollution.

The Process of Biological Nitrogen Fixation

• Biological Nitrogen Fixation (BNF) is the conversion of N2 gas to ammonia.
• 941 \text{ kJ/mol} of energy is required to break the bond in N2.

Nitrogenase Enzyme

• The nitrogenase enzyme is a complex protein formed by many genes.
• Nif genes are a cluster of genes required for BNF.
• Each organism with Nif genes has a unique combination.

Symbiotic BNF

• Symbiotic BNF occurs between legumes and bacteria (rhizobia).
• Roots are infected and colonized by symbiotic rhizobia through inter-specific signaling.
• This leads to changes in gene expression (transcription).
• Both the plant host and rhizobia undergo developmental changes and changes in physiology (phenotype).
• Atmospheric N is fixed into usable ammonium.

Biochemical Interactions

• Legumes can distinguish rhizobia from other bacteria through specific biochemical interactions.
• Process:
  • Rhizobia interact with root hairs.
  • Nod factors are released.
  • Infection threads form.
  • Root nodules develop.
  • Nitrogenase converts N2 + 8H^+ \rightarrow 2NH3 + H_2.

Rhizobia and Sugar Exchange

• Rhizobia receive sugar (malate) in exchange for fixing nitrogen for the host plant.
• This requires energy and involves the nif & fix genes.
• Flavonoids signal between the plant and rhizobia, with nod genes playing a role.

Nodule Environment

• The nodule provides a perfect environment for BNF.
  • N-fixing rhizobia are present in the nodule.
  • Bacteroids.
  • Bacteroids are surrounded by a membrane impermeable to oxygen.

Oxygen Sensitivity

• Nitrogen fixation occurs only in the absence of oxygen because the nitrogenase metal cluster is prone to oxidation.
• The membrane in the symbiosome is impermeable to oxygen.

Energy Requirements

• Nitrogen fixation is an energy-intensive process.
• 16 \text{ ATP} are required for every molecule of N2 fixed by nitrogenase.
• ATP comes from photosynthesis-derived sugars.
• Synthetic nitrogen fixation (Haber-Bosch process):
  • Requires 450-500 ^\circ C and pressures up to 150-300 \text{ atm}.
  • Consumes 20-30 \text{ kWh/kg} ammonia.
  • Example: A fridge uses 1-2 \text{ kWh/day}.
  • 1 \text{ million tonnes} of nitrogen are sold in Australia annually (2002-2017).

Regulation of Nodules

• Nodule number is regulated by the host plant based on the need for nitrogen (N) vs. energy expense (C).
• Supernodulation mutants exist, contrasting with wild-type plants.

Molecular Control

• Legumes control symbiosis through:
  1. Autoregulation of nodulation (number of rhizobia/nodules).
  2. Nitrogen regulation of nodulation (based on available nitrogen in the soil).

Autoregulation of Nodulation

• After the first nodules begin forming:
  • Changes in gene expression occur.
  • Small peptides are produced and travel to the shoot.
  • Perceived by a receptor (NARK receptor).
  • Information flows back to the root to inhibit further nodulation.

NARK Gene Mutations

• The pathway functions to prevent a beneficial symbiosis from becoming parasitic.
• Two known gene mutations:
  • Mutation in LRR domain (G>E) changes secondary structure.
  • Mutation in Kinase domain (W>[star]) renders it unable to catalyze phosphorylation.
  • LRR, SP, TM, Kinase domains are affected.
  • Mutations can result in supernodulation phenotypes.

Abiotic Factors

• Abiotic factors can change the symbiosis and biological nitrogen fixation, including:
  • Precipitation
  • Soil type
  • pH
  • Nutrients (P, Fe)
  • Temperature
  • UVR (UV Radiation)
  • Geographical factors
  • CO_2 concentration

Nitrate Inhibition

• Increased nitrate concentration (e.g., 2.5 \text{ mM } KNO3, 10 \text{ mM } KNO3) decreases nitrogenase activity and nodule number.
• Other nitrogen forms (urea, ammonium nitrate) also inhibit nodulation.

Mycorrhizal Symbiosis

• Different microbial symbioses help plants acquire other essential nutrients.
• Insoluble soil phosphates are made available to plants by arbuscular mycorrhizal fungi.
• These fungi secrete enzymes and acids to solubilize phosphate (phosphatases, organic acids).
• The symbiosis provides host plants with P and other nutrients in exchange for photosynthesis-derived sugars (for ATP).
• The symbiosis triggers gene expression changes, but the plant does not produce new organs.

Nutritional Specialists

• Different specialists include:
  • Ericoid (1.4%)
  • Ectomycorrhizal (2%)
  • Cluster roots
  • Carnivores
  • Parasites
  • Epiphytes
  • Arctic alpine
  • Aquatic or marine
  • Halophytes
  • Arid
  • Non-mycorrhizal (NM) (8%)
  • Orchid (10%)
  • NM-AM (7%)
  • Arbuscular mycorrhiza (AM) (71%)

Applications in Agriculture

• Legumes are integrated into many management systems to:
  • Gain N content of soil.
  • Lower synthetic fertilizer input.
  • Intercropping.
  • Crop rotation.
  • Green and brown manuring.
  • Cover cropping for added N for subsequent crops.

Inoculating Legumes

• Inoculating legumes decreases flowering time.
• Examples include:
  • Glycine max
  • Arachis hypogaea
  • Medicago sativa
  • Lotus japonicus
  • Unino. and Ino treatments show significant differences (P<0.0001).

Future Applications

• Current Method: Haber-Bosch Process
  • N2 + H2 \rightarrow NH_3
  • Requires: 400^\circ C & \text{>150 bar}
• Future Method: Nitrogenase Bioreactor
  • N2 \rightarrow NH3 + H_2
  • Requires: 30^\circ C & \text{1 bar}
  • Involves: Microbiology, yeast, and biochemistry.
• Requires an interdisciplinary approach: Plant biology, microbiology, biochemistry, synthetic biology/bioinformatics.

Environmental Importance

• Legumes are vital in supporting natural ecosystems by improving the soil.

Summary

• Biological Nitrogen Fixation is the process of converting atmospheric nitrogen to plant-available forms.
• The reaction is catalyzed by the bacterial nitrogenase enzyme.
• Legumes form a symbiosis with rhizobia, called nodulation.
• Legumes have genetic controls and biochemical pathways to prevent the symbiosis from becoming detrimental.
• BNF and nodulation effectiveness are determined by abiotic influences.
• Other symbioses occur to help the plant obtain other important nutrients.
• It's a useful biochemical process for agriculture and natural ecosystems.