NITROGEN FIXATION: BIOTIC AND ABIOTIC PROCESSES - BIOLOGY - 2025

The nitrogen fixation is the set of biological and non - biological processes which produce chemical forms of nitrogen available to living things. Nitrogen availability controls in an important way the functioning of ecosystems and global biogeochemistry, since nitrogen is a factor that limits the net primary productivity in terrestrial and aquatic ecosystems.

In the tissues of living organisms, nitrogen is part of amino acids, units of structural and functional proteins such as enzymes. It is also an important chemical element in the constitution of nucleic acids and chlorophyll.

Additionally, the biogeochemical reactions of carbon reduction (photosynthesis) and carbon oxidation (respiration), occur through the mediation of nitrogen-containing enzymes, since they are proteins.

In the chemical reactions of the biogeochemical nitrogen cycle, this element changes its oxidation states from zero in N _2, to 3- in NH ₃, 3+ in NO ₂ ^- and NH ₄ ⁺, and to 5+ in NO ₃ ^-.

Several microorganisms take advantage of the energy generated in these nitrogen oxide-reduction reactions and use it in their metabolic processes. It is these microbial reactions that collectively drive the global nitrogen cycle.

The most abundant chemical form of nitrogen on the planet is gaseous molecular diatomic nitrogen N ₂, which constitutes 79% of the Earth's atmosphere.

It is also the least reactive nitrogen chemical species, practically inert, very stable, due to the triple bond that joins both atoms. For this reason, the abundant nitrogen in the atmosphere is not available to the vast majority of living beings.

Nitrogen in chemical forms available to living beings is obtained through "nitrogen fixation." Nitrogen fixation can occur through two main ways: abiotic forms of fixation and biotic forms of fixation.

Abiotic forms of nitrogen fixation

Electric storms

Figure 2. Electrical storm Source: pixabay.com

The lightning or "lightning" produced during electrical storms is not just noise and light; they are a powerful chemical reactor. Due to the action of lightning, nitrogen oxides NO and NO ₂ are produced during storms, generically called NO _x.

These electrical discharges, observed as lightning bolts, generate conditions of high temperatures (30,000 ^o C) and high pressures, which promote the chemical combination of oxygen O ₂ and nitrogen N ₂ from the atmosphere, producing nitrogen oxides NO _x.

This mechanism has a very low rate of contribution to the total rate of nitrogen fixation, but it is the most important among abiotic forms.

Burns fossil fuels

There is an anthropogenic contribution to the production of nitrogen oxides. We have already said that the strong triple bond of the nitrogen molecule N ₂ can only be broken under extreme conditions.

The combustion of fossil fuels derived from petroleum (in industries and in commercial and private transportation, maritime, air and land), produces enormous amounts of NO _x emissions into the atmosphere.

The N ₂ O emitted in the combustion of fossil fuels is a powerful greenhouse gas that contributes to global warming of the planet.

Biomass burning

There is also a contribution of nitrogen oxides NO _x by burning biomass in the area with the highest temperature of the flame, for example in forest fires, use of firewood for heating and cooking, incineration of organic garbage and any use of biomass as a source of caloric energy.

Nitrogen oxides NOx emitted into the atmosphere by anthropogenic routes cause serious environmental pollution problems, such as photochemical smog in urban and industrial environments, and important contributions to acid rain.

Nitrogen emissions from soil erosion and rock weathering

Soil erosion and nitrogen-rich bedrock weathering expose minerals to the elements that can release nitrogen oxides. Bedrock weathering occurs due to exposure to environmental factors, caused by physical and chemical mechanisms acting together.

Tectonic movements can physically expose nitrogen-rich rocks to the elements. Subsequently, by chemical means, the acid rain precipitation causes chemical reactions that release NO _x, both from this type of rocks and from the soil.

There is recent research that assigns 26% of the planet's total bioavailable nitrogen to these mechanisms of soil erosion and rock weathering.

Biotic forms of nitrogen fixation

Some bacterial microorganisms have mechanisms capable of breaking the triple bond of N ₂ and producing ammonia NH ₃, which is easily transformed into ammonium ion, metabolizable NH ₄ ⁺.

Free-living or symbiotic microorganisms

The forms of nitrogen fixation by microorganisms can occur through free-living organisms or through organisms that live in symbiotic associations with plants.

Although there are great morphological and physiological differences between nitrogen-fixing microorganisms, the fixation process and the nitrogenase enzyme system used by all of these are very similar.

Quantitatively, biotic nitrogen fixation through these two mechanisms (free life and symbiosis) is the most important globally.

Mechanisms to keep the nitrogenase system active

Nitrogen-fixing microorganisms have strategic mechanisms to keep their nitrogenase enzymatic system active.

These mechanisms include respiratory protection, conformational chemical protection, reversible inhibition of enzyme activity, additional synthesis of an alternative nitrogenase with vanadium and iron as cofactors, creation of diffusion barriers for oxygen, and spatial separation of nitrogenase.

Some have microaerophilia, such as the chemotrophic bacteria of the genera Azospirilium, Aquaspirillum, Azotobacter, Beijerinkia, Azomonas, Derxia, Crynebacterium, Rhizobium, Agrobacterium, Thiobacillus, and phototrophs of the genera Gleocapsa, Anabaena, Lingcillatoria, Spirulina, Spirulina, and Lingcillatoria, Spirultoria, Spirulina, and Nostrum.

Others present facultative anaerobiosis, such as the chemotrophic genera: Klebsiella, Citrobacter, Erwinia, Bacillus, Propionibacterium and phototrophs of the Rhodospirillum, Rhodopsuedomonas genera.

Biotic nitrogen fixation by free-living microorganisms

Nitrogen-fixing microorganisms that live in the soil in a free (asymbiotic) form are basically archaebacteria and bacteria.

There are several types of bacteria and cyanobacteria that can convert atmospheric nitrogen, N _2, to ammonia, NH ₃. According to the chemical reaction:

N ₂ + 8H ⁺ + 8e ^- +16 ATP → 2 NH ₃ + H ₂ +16 ADP + 16Pi

This reaction requires the mediation of the nitrogenase enzyme system and a cofactor, vitamin B ₁₂. Additionally, this nitrogen fixation mechanism consumes a lot of energy, is endothermic and requires 226 Kcal / mol of N ₂; In other words, it carries a high metabolic cost, which is why it must be coupled to a system that produces energy.

Energy required during the N-fixation reaction

The energy for this process is obtained from ATP, which comes from oxidative phosphorylation coupled to the electron transport chain (which uses oxygen as the final electron acceptor).

The process of reducing molecular nitrogen to ammonia also reduces hydrogen in proton form H ⁺ to molecular hydrogen H _2.

Many nitrogenase systems have coupled a hydrogen recycling system mediated by the hydrogenase enzyme. Nitrogen-fixing cyanobacteria couple photosynthesis to nitrogen fixation.

The enzyme complex nitrogenase and oxygen

The nitrogenase enzyme complex has two components, component I, dinitrogenase with molybdenum and iron as cofactors (which we will call Mo-Fe-protein), and component II, dinitrogenase reductase with iron as cofactor (Fe-protein).

The electrons involved in the reaction are donated first to component II and later to component I, where the nitrogen reduction occurs.

For the transfer of electrons from II to I to occur, the Fe-protein is required to bind to a Mg-ATP at two active sites. This union generates a conformational change in the Fe-protein. An excess of oxygen can produce another unfavorable conformational change in the Fe-protein, since it cancels its electron-accepting capacity.

This is why the nitrogenase enzyme complex is very susceptible to the presence of oxygen above tolerable concentrations and that some bacteria develop microaerophilic life forms or facultative anaerobiosis.

Among the free-living nitrogen-fixing bacteria, we can mention the chemotrophs belonging to the genera Clostridium, Desulfovibrio, Desulfotomaculum, Methanosarcina, and the phototrophs of the genera Chromatium, Thiopedia, Ectothiordospira, among others.

Biotic nitrogen fixation by microorganisms of symbiotic life with plants

There are other nitrogen-fixing microorganisms that are capable of establishing symbiotic associations with plants, particularly with legumes and grasses, either in the form of ectosymbiosis (where the microorganism is located outside the plant), or endosymbiosis (where the microorganism lives within cells or intercellular spaces of the plant).

Most of the nitrogen fixed in terrestrial ecosystems comes from the symbiotic associations of bacteria of the genera Rhizobium, Bradyrhizobium, Sinorhizobium, Azorhizobium, Allorhizoium and Mesorhizobium, with legume plants.

There are three interesting types of nitrogen-fixing symbioses: associative rhizocenoses, systems with cyanobacteria as symbionts, and mutualistic endorizobioses.

Rhizocenosis

In associative rhizocenosis-like symbioses, specialized structures are not formed in the roots of plants.

Examples of this type of symbiosis are established between corn (Zea maiz) and sugar cane (Saccharum officinarum) plants with Gluconacetobacter, Azoarcus, Azospirillum and Herbaspirillum.

In rhizocenosis, the nitrogen-fixing bacteria uses the root exudate of the plant as a nutritive medium and colonizes the intercellular spaces of the root cortex.

Symbiotic cyanobacteria

In systems where cyanobacteria participate, these microorganisms have developed special mechanisms for the coexistence of anoxic nitrogen fixation and their oxygenic photosynthesis.

For example, in Gleothece and Synechococcus, they temporarily separate: they perform daytime photosynthesis and nighttime nitrogen fixation.

In other cases, there is spatial separation of both processes: nitrogen is fixed in groups of differentiated cells (heterocysts), where photosynthesis does not take place.

Nitrogen-fixing symbiotic associations of cyanobacteria of the genus Nostoc have been studied with non-vascular plants (antóceras), as in cavities of Nothocerus endiviaefolius, with liverworts Gakstroemia magellanica and Chyloscyphus obvolutus in ectosymbiosis separately, with bryophytes (forming rhizoids in mosquens), and with higher angiosperm plants, for example with the 65 perennial herbs of the genus Gunnnera.

For example, the symbiotic nitrogen-fixing association of cyanobacteria Anabaena with a bryophyte, a non-vascular plant, has been observed in leaves of the small fern Azolla anabaenae.

Endorhizobiosis

As examples of endorhizobiosis, we can cite the association called actinorrhiza that is established between Frankia and some woody plants such as casuarina (Casuarina cunninghamiana) and alder (Alnus glutinosa), and the Rhizobium –leguminous association.

Most of the species of the Leguminosae family form symbiotic associations with Rhizobium bacteria and this microorganism has evolutionary specialization in transferring nitrogen to the plant.

In the roots of plants associated with Rhizobium, so-called radical nodules appear, where nitrogen fixation takes place.

In Sesbania and Aechynomene legumes, additional nodules are formed on the stems.

• Chemical signals

There is an exchange of chemical signals between the symbiont and the host. Plants have been found to exude certain types of flavonoids that induce the expression of nod genes in Rhizobium, which produce nodulation factors.

The nodulation factors generate modifications in the root hairs, formation of an infection channel and cell division in the root cortex, which promote the formation of the nodule.

Some examples of nitrogen-fixing symbiosis between higher plants and microorganisms are shown in the following table.

Mycorrhizobiosis

Additionally, in most ecosystems, there are nitrogen-fixing mycorrhizal fungi, belonging to the phyla Glomeromycota, Basidiomycota and Ascomycota.

Mycorrhizal fungi can live in ectosymbiosis, forming a hyphal sheath around the fine roots of some plants and spreading additional hyphae throughout the soil. Also in many tropical areas, plants host mycorrhizae in endosymbiosis, the hyphae of which penetrate root cells.

It is possible that a fungus forms mycorrhizae with several plants simultaneously, in which case interrelationships are established between them; or that the mycorrhizal fungus is parasitized by a plant that does not photosynthesize, mycoheterotrophic, such as those of the genus Monotropa. Also several fungi can establish symbiosis with a single plant simultaneously.

References

1. Inomura, K. , Bragg, J. and Follows, M. (2017). A quantitative analysis of the direct and indirect costs of nitrogen fixation. The ISME Journal. 11: 166-175.
2. Masson-Bovin, C. and Sachs, J. (2018). Symbiotic nitrogen fixation by rhizobia - the roots of a success story. Plant Biology. 44: 7-15. doi: 10.1016 / j.pbi.2017.12.001
3. Menge, DNL, ​​Levin, SA and Hedin, LO (2009). Facultative versus obligate nitrogen fixation strategies and their ecosystem consequences. The American Naturalist. 174 (4) doi: 10.1086 / 605377
4. Newton, WE (2000). Nitrogen fixation in perspective. In: Pedrosa, FO Editor. Nitrogen fixation from molecules to crops productivity. Netherlands: Kluwer Academic Publishers. 3-8.
5. Pankievicz; VCS, do Amaral; FP, Santos, KDN, Agtuca, B., Xu, Y., Schultes, MJ (2015). Robust biological nitrogen fixation in a model grass-bacterial association. The Plant Journal. 81: 907-919. doi: 10.1111 / tpj.12777.
6. Wieder, WR, Cleveland, CC, Lawrence, D. and Bonau, GB (2015). Effects of model structural uncertainty on carbon cycle projections: biological nitrogen fixation as a case of study. Environmental Research Letters. 10 (4): 1-9. doi: 10.1088 / 1748-9326 / 10/4/044016