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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.