Biological Nitrogen Fixation Notes

Biological Nitrogen Fixation (BNF) is a crucial part of the nitrogen cycle.
The phyllosphere, rhizosphere, endosphere, and bulk soil all contain unique microbiomes that interact with plants.

Involves detailed biochemical pathways in both the nitrogen-fixing bacteria (rhizobia) and the host plant (legume).
Abiotic factors influence the symbiosis.
Another symbiosis to consider is mycorrhizae.

The atmosphere is composed of 78% N2.
N2 is reduced to NH3 (ammonia) through:
- Biological Nitrogen Fixation (BNF) by bacteria.
- Industrial N2 fixation.
Nitrogen-containing fertilizers supply plants with nitrogen.
N assimilation occurs within plants.
Excess NH3 is processed by microorganisms through nitrification and denitrification.
These processes can cause environmental pollution.

BNF is the conversion of N2 gas to ammonia (NH3).
It requires a significant amount of energy to break the N2 triple bond: $941 \, \text{kJ/mol}$ .

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

Legumes can distinguish rhizobia from other bacteria through specific biochemical interactions.
Key components of the interaction include:
- Flavonoids: produced by the plant to attract rhizobia.
- Nod factors: signaling molecules produced by rhizobia.
- Infection thread: a structure that allows bacteria to invade root cells.
Nitrogenase facilitates the conversion of dinitrogen gas and protons into 2 molecules of ammonia and one molecule of hydrogen gas: $N<em>2 + 8H^+ \xrightarrow{\text{nitrogenase}} 2NH</em>3 + H_2$

The nodule provides the perfect environment for BNF.
- N-fixing Rhizobia reside within the nodule.
- Rhizobia differentiate into bacteroids.
- Bacteroids are surrounded by a membrane impermeable to oxygen.

Nitrogen fixation occurs only in the absence of oxygen.
The nitrogenase metal cluster is prone to oxidation.
The symbiosome membrane is impermeable to oxygen, maintaining an anaerobic environment.

Nitrogen fixation is an energy-intensive process.
$16 \text{ ATP}$ are required for every molecule of N2 fixed by nitrogenase.
ATP is derived from photosynthesis-derived sugars.
Synthetic nitrogen fixation uses the Haber-Bosch process.
- Requires high temperatures ( $450-500 \,^{\circ}\text{C}$ ) and pressures (up to $150-300 \, \text{atm}$ ).
- Consumes significant energy: $20-30 \, \text{kWh/kg}$ ammonia.
One million tonnes of nitrogen were sold annually in Australia between 2002-2017.

The host plant regulates nodule number to balance nitrogen needs and energy expenditure.
Supernodulation mutants exist.

Legumes have molecular mechanisms to control the symbiosis:
1. During nodulation, based on the number of rhizobia/nodules present (Autoregulation of Nodulation).
2. Before symbiosis, based on available nitrogen in the soil (Nitrogen regulation of nodulation).

The NARK pathway prevents a beneficial symbiosis from becoming parasitic.
Two known gene mutations:
- Mutation in LRR domain (G>E) changes secondary structure.
- Mutation in Kinase domain (W> *) renders it unable to catalyze phosphorylation.
Specific mutations and their locations:
- G225E in LRR domain
- W791* in Kinase domain

High nitrate concentrations (e.g., $2.5 \, \text{mM KNO}<em>3$ or $10 \, \text{mM KNO}</em>3$ ) can decrease nitrogenase activity and nodule number.
Other nitrogen forms (urea, ammonium nitrate) also inhibit nodulation.

Different microbial symbioses help plants acquire other essential nutrients.
Arbuscular mycorrhizal fungi solubilize insoluble soil phosphates by secreting:
- Phosphatases
- Organic acids
Symbiosis provides host plants with P and other nutrients in exchange for photosynthesis-derived sugars.
Symbiosis triggers gene expression changes, but the plant does not produce new organs.

Legumes are integrated into many agricultural management systems to:
- Increase soil nitrogen content.
- Lower synthetic fertilizer input.
- Examples include Intercropping, Crop rotation, Green, brown manuring and Cover cropping.
Added N benefits the subsequent crop.

Inoculation decreases the time for flowering in various legumes, as demonstrated in studies involving:
- Glycine max
- Arachis hypogaea
- Lotus japonicus
- Medicago sativa

Microbiology, yeast and biochemistry will be used in future applications.
Use plant biology, microbiology, synthetic biology/bioinformatics and biochemistry to convert N2 to NH3.

Process of converting atmospheric nitrogen to plant-available forms.
Reaction 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.
Useful biochemical process for agriculture and natural ecosystems.

How are photosynthesis and nodulation linked? Include details of their biochemical pathways.
Briefly describe the symbiosis between plants and mycorrhizae in a biochemical framework.
Compare and contrast biological nitrogen fixation by legumes and nitrogen uptake by non-legumes.