Introduction

Nitrogen is essential for life because it forms part of proteins, nucleic acids and many other cellular compounds. Although approximately 78% of Earth's atmosphere consists of nitrogen gas (N₂), most organisms cannot use this abundant resource directly. Plants generally absorb nitrogen from soil in forms such as ammonium and nitrate, while animals obtain it through food. The difficulty lies in the chemical stability of atmospheric nitrogen. Its two nitrogen atoms are connected by a strong triple bond, making the molecule relatively unreactive under ordinary biological conditions. Certain microorganisms have evolved a remarkable biochemical solution to this problem: they use a specialized enzyme system called nitrogenase to convert atmospheric nitrogen into ammonia.

This process, known as biological nitrogen fixation, is carried out by microorganisms called diazotrophs. Nitrogen-fixing bacteria and archaea occur in soils, aquatic environments, plant-associated communities and oxygen-limited habitats. Their activities introduce biologically available nitrogen into ecosystems, supporting microbial growth, plant nutrition and broader nutrient cycling. Research has traditionally emphasized nitrogen-fixing bacteria associated with legume roots, but recent discoveries have revealed additional mechanisms of nitrogen fixation in marine microorganisms and archaea. Scientists have also characterized a nitrogen-fixing organelle in a marine alga, providing an unusual example of how microbial partnerships can become integrated into eukaryotic cells.

What Is Biological Nitrogen Fixation?

Biological nitrogen fixation is the enzyme-catalysed conversion of atmospheric nitrogen gas into ammonia. The reaction requires electrons, energy and specialized proteins containing metal–sulfur clusters. In the best-characterized molybdenum-dependent nitrogenase system, the overall reaction requires at least 16 molecules of ATP for each molecule of N₂ reduced, producing two molecules of ammonia and one molecule of hydrogen gas. The actual energy expenditure of a cell may be greater because additional energy is needed to generate reducing power, maintain metabolism and protect the nitrogenase system from damaging conditions.

The ammonia produced can be incorporated into microbial metabolism, usually through ammonium-dependent assimilation pathways. Microorganisms use this nitrogen to synthesize amino acids, nucleotides and other essential cellular components. In symbiotic relationships, some fixed nitrogen becomes available to the host organism, while free-living diazotrophs contribute to nutrient cycling through microbial growth, release of nitrogen-containing compounds and decomposition. Nitrogen fixation is therefore one of the processes that connect atmospheric nitrogen to the biological nitrogen cycle.

Nitrogen fixation should not be confused with nitrification or denitrification. Nitrification involves the oxidation of reduced nitrogen compounds, while denitrification can convert oxidized nitrogen compounds into gaseous products, including N₂. These processes involve different microorganisms, enzymes and environmental conditions. Together, they form part of a complex network of microbial nitrogen transformations rather than a single, fixed sequence of reactions.

How Does the Nitrogenase Enzyme Work?

Nitrogenase is the enzyme system responsible for biological nitrogen fixation. The most extensively studied form is molybdenum nitrogenase, which consists of two major protein components. The iron protein, encoded by nifH, transfers electrons using energy from ATP hydrolysis. The molybdenum–iron protein, formed by NifD and NifK subunits, contains the catalytic machinery where nitrogen gas is reduced to ammonia. Electron transfer between these components involves metal–sulfur clusters that allow the enzyme to carry out a chemically demanding reaction under biological conditions.

The reduction of N₂ requires a series of coordinated electron and proton transfers. Although nitrogenase has been investigated for decades, several details of its catalytic mechanism remain subjects of active research. Alternative nitrogenase systems containing vanadium or iron-only catalytic components also occur in some microorganisms. These systems demonstrate that nitrogen fixation is not limited to a single metal composition, although molybdenum nitrogenase remains the best-characterized example.

One of the greatest challenges for nitrogen-fixing organisms is the sensitivity of nitrogenase to oxygen. Oxygen can damage the metal-containing components of the enzyme, making protection essential for sustained activity. Different microorganisms have developed different solutions. Some fix nitrogen under anaerobic conditions, while others maintain low intracellular oxygen concentrations through respiratory activity, specialized cellular structures or separation of nitrogen fixation from oxygen-producing processes. These strategies illustrate how nitrogenase activity depends not only on the enzyme itself but also on the physiology of the organism.

Which Microorganisms Can Fix Nitrogen?

Nitrogen fixation is distributed among diverse bacteria and archaea. Some bacteria, such as Azotobacter vinelandii, can fix nitrogen while living freely in soil. Certain cyanobacteria, including members of Trichodesmium, contribute to nitrogen fixation in aquatic environments. Other diazotrophs form associations with plants or other microorganisms, where they may exchange fixed nitrogen for carbon compounds or other resources. The ecological roles of these organisms vary according to their metabolic capabilities and the environments they inhabit.

Nitrogen-fixing archaea are particularly interesting because they demonstrate that this metabolism extends beyond bacteria. Some methanogenic archaea possess functional nitrogenase systems and carry out nitrogen fixation in oxygen-limited environments. Although their nitrogenases share fundamental catalytic features with bacterial systems, differences in protein organization and regulation may reveal additional ways of controlling the process. Importantly, nitrogen fixation is not a universal characteristic of any broad microbial group. Its occurrence depends on the presence of functional nitrogenase machinery and suitable physiological conditions.

How Do Rhizobia Form Nitrogen-Fixing Partnerships with Plants?

One of the most familiar examples of biological nitrogen fixation occurs in the symbiosis between legumes and rhizobia. Plants such as peas, beans and clover can develop specialized root structures called nodules that accommodate compatible bacteria. Within these nodules, the plant supplies carbon compounds and maintains conditions that support bacterial metabolism, while the bacteria fix atmospheric nitrogen. This relationship can contribute substantially to plant nitrogen nutrition and is important in agricultural systems where legumes are cultivated.

The formation of nitrogen-fixing nodules requires communication between the plant and its bacterial partner. Legume roots release compounds, including flavonoids, that can activate the bacterial regulatory protein NodD. Activated NodD stimulates genes involved in producing Nod factors, which are signalling molecules recognized by compatible host plants. This recognition initiates developmental responses associated with bacterial infection and nodule formation. Because plants and bacteria differ in their signalling systems, many rhizobial partnerships show host specificity.

In January 2026, Ruan and colleagues published research examining the molecular basis of NodD activation by flavonoids. Using structural and functional approaches, the researchers investigated NodD from Rhizobium leguminosarum and compared its recognition properties with those of NodD1 from Sinorhizobium medicae. Their findings identified structural features that influence how these regulatory proteins respond to different flavonoids. The researchers also demonstrated that modifying selected recognition features could alter signalling behaviour and restore symbiotic function in a particular experimental mutant system.

The study provides important insight into the molecular communication underlying legume–rhizobium symbiosis. However, modifying NodD alone would not necessarily enable any bacterium to form an effective nitrogen-fixing partnership with any plant. Successful symbiosis requires several additional processes, including compatible infection, nodule development, nitrogenase activity and appropriate exchange of nutrients between the partners.

How Do Plants Regulate Their Nitrogen-Fixing Partners?

The legume–rhizobium relationship is often described as mutually beneficial, but not all bacterial strains contribute equally to nitrogen fixation. A plant may host several rhizobial strains, some of which provide more fixed nitrogen than others. Since maintaining nodules requires resources, plants can benefit from regulating the amount of carbon and other nutrients supplied to less effective partners.

Underwood and Poole investigated this process in pea plants using closely related Rhizobium leguminosarum strains with different nitrogen-fixing abilities. Their study, published online in April 2026, included split-root experiments designed to examine whether the plant responds to individual nodules independently or evaluates their performance in relation to other nodules on the same root system. The researchers found that pea plants could distinguish relatively small differences in bacterial effectiveness and that resource allocation depended partly on the overall composition of the root system.

An interesting finding was that less effective rhizobial strains could sometimes avoid reduced resource allocation when they dominated nodulation. This suggests that plant regulation is influenced by the relative performance of available symbiotic partners rather than operating through an identical response to every ineffective nodule. The results improve our understanding of how legumes manage microbial partnerships, although they were obtained in a defined pea–rhizobium experimental system and should not automatically be generalized to all plant–microbe associations.

Nitrogen Fixation in Marine Environments

Marine nitrogen fixation has often been associated with cyanobacteria living in warm, nutrient-poor surface waters. However, molecular studies have revealed a much wider diversity of potential nitrogen-fixing microorganisms, including non-cyanobacterial diazotrophs. Their ecological roles remain difficult to determine because the presence of nitrogenase genes does not necessarily mean that nitrogen fixation is actively occurring.

In March 2026, Turk-Kubo and colleagues reported research investigating nitrogen-fixing organisms in the North Sea and Norwegian coastal waters. The fieldwork was conducted during July 2023 and combined measurements of nitrogen-fixation activity with molecular investigations of microbial populations. The researchers detected the Braarudosphaera/UCYN-A association and measured nitrogen-fixation rates reaching approximately 110 nanomoles of nitrogen per liter per day in the investigated waters. Based on their measurements and calculations, they estimated that nitrogen fixation could support approximately 4-24% of summertime net primary production in the studied region.

These findings suggest that nitrogen fixation may contribute more to productivity in temperate shelf waters than previously recognized. However, the measured rates and estimated contribution apply to the investigated locations and sampling period. The simultaneous detection of Braarudosphaera/UCYN-A and nitrogen-fixation activity also does not demonstrate that this partnership was responsible for all the activity measured in the samples.

What Is a Nitroplast?

One of the most unusual discoveries in nitrogen-fixation research concerns the marine alga Braarudosphaera bigelowii and its association with a nitrogen-fixing cyanobacterial lineage historically known as UCYN-A. This relationship was originally studied as an example of endosymbiosis, in which one organism lives within another. However, research has revealed an unusually high degree of integration between the algal host and its nitrogen-fixing partner.

In 2024, Coale and colleagues reported evidence that the nitrogen-fixing structure associated with the studied Braarudosphaera lineage possesses characteristics of a cellular organelle. Using cellular imaging and proteomic analysis, the researchers demonstrated that the structure divides in coordination with the host cell and receives proteins encoded by the algal genome. These observations support the interpretation that the former cyanobacterial endosymbiont has become integrated into the host's cellular organization. The researchers described this nitrogen-fixing structure as a nitroplast.

The discovery is important for understanding organelle evolution. Mitochondria and chloroplasts also originated through ancient endosymbiotic relationships, although their evolutionary histories are distinct. The nitroplast provides a comparatively recent example of how a microbial symbiont can become an integrated cellular component. Nevertheless, the strongest evidence concerns the investigated Braarudosphaera–UCYN-A lineage, particularly UCYN-A2. It should not be assumed that every related association has undergone the same degree of integration.

Can Marine Particles Support Nitrogen Fixation?

Many marine microorganisms live on or within particles composed of organic material, including aggregates formed from phytoplankton and other biological substances. These particles can develop local differences in oxygen concentration, nutrient availability and microbial activity. Their interiors may provide favourable conditions for certain nitrogen-fixing microorganisms, particularly when oxygen consumption creates microenvironments that reduce exposure to oxygen.

Reeder and colleagues investigated non-cyanobacterial diazotrophs associated with suspended and sinking particles in the Baltic Sea. Their study, published online in August 2026, used nifH gene amplicon sequencing to examine microbial communities across different phases of primary productivity. Non-cyanobacterial sequences accounted for a large proportion of the recovered diazotroph-associated sequences, and community composition changed between productivity phases. These findings indicate that particle-associated environments contain diverse microorganisms with genetic potential for nitrogen fixation. However, the proportions of recovered gene sequences should not be interpreted as direct measurements of nitrogen-fixation activity.

Further experimental evidence was reported by Coët and colleagues in September 2026. The researchers investigated Vibrio diazotrophicus colonizing laboratory-generated diatom aggregates and combined nitrogenase protein detection, cellular respiration measurements and nitrogen-15 tracing. Their results showed that the spatial distribution of bacterial cells within particles was associated with conditions favourable for nitrogen fixation. Nitrogenase-expressing cells were relatively more abundant toward particle interiors under the investigated conditions, supporting the idea that marine particles can provide specialized microenvironments for heterotrophic diazotrophs.

These findings help explain why microbial activity within a particle may differ from activity in the surrounding water. However, the experiments used a controlled model system, and the results do not establish that every natural marine particle supports nitrogen fixation at the same rate or through the same mechanisms.

How Do Microorganisms Control Nitrogenase Activity?

Nitrogen fixation requires substantial energy, so microorganisms must regulate the process according to their metabolic requirements and the availability of nitrogen-containing compounds. When suitable fixed nitrogen is already available, continued nitrogenase activity may impose an unnecessary energetic cost. Regulation can therefore occur through changes in gene expression, protein activity and interactions between nitrogenase and other regulatory proteins.

In October 2026, Kashyap and colleagues published a study in Nature investigating nitrogenase regulation in the methanogenic archaeon Methanosarcina acetivorans. Using cryo-electron microscopy, the researchers determined the structure of a large nitrogenase–PII protein supercomplex. The structure contained three NifDK catalytic units associated with six regulatory PII protein assemblies. These regulatory proteins prevented normal interaction between the catalytic nitrogenase component and the NifH electron-transfer protein, maintaining the enzyme in an inactive configuration.

The researchers also investigated how metabolic signals influence the complex. In laboratory experiments, adding 2-oxoglutarate and ATP promoted the release of regulatory proteins and increased measured nitrogenase activity approximately threefold in vitro. These findings provide structural and biochemical evidence that nitrogenase activity in this archaeon can be controlled through direct interactions between the enzyme and regulatory proteins. However, the mechanism was demonstrated in a particular methanogenic system and should not be assumed to operate identically in all nitrogen-fixing organisms. The reported increase in enzyme activity also does not represent an improvement in agricultural nitrogen fixation.

Can Nitrogenase Function at Extremely High Temperatures?

Some nitrogen-fixing microorganisms inhabit environments where temperatures are far above those tolerated by most familiar bacteria. Studying these organisms can reveal how enzymes maintain their structure and activity under extreme conditions. In September 2026, Maslać and colleagues reported research on the hyperthermophilic archaeon Methanocaldococcus infernus, investigating its ability to grow using atmospheric nitrogen at temperatures approaching 92°C.

The researchers purified the organism's nitrogenase and investigated its biochemical and structural properties. Their findings demonstrated unusual thermal stability and provided high-resolution structural information about features associated with adaptation to elevated temperatures. The study expands our understanding of the environmental conditions under which biological nitrogen fixation can occur and shows that nitrogenase systems can function in organisms adapted to extreme habitats.

However, the discovery of a highly thermostable nitrogenase does not automatically establish its suitability for industrial ammonia production. Practical applications would require further evaluation of enzyme activity, energy requirements, stability under processing conditions and compatibility with other components of a production system.

How Do Scientists Measure Biological Nitrogen Fixation?

One of the most important challenges in nitrogen-fixation research is distinguishing the genetic potential to perform the process from evidence that it is actively occurring. The nifH gene, which commonly encodes the nitrogenase iron protein, is widely used as a molecular marker for investigating diazotroph diversity. Detecting nifH sequences can reveal microorganisms that potentially possess nitrogen-fixing machinery, but gene detection alone does not demonstrate that the genes are expressed or that nitrogen fixation is taking place.

Researchers therefore combine molecular investigations with activity measurements. One established approach involves incubating samples with nitrogen gas enriched in the stable isotope nitrogen-15 and measuring its incorporation into biological material. This provides evidence that nitrogen derived from N₂ has entered the sampled biological system. Another widely used technique is the acetylene reduction assay, which measures the conversion of acetylene into ethylene as an indicator of nitrogenase activity. However, converting acetylene reduction measurements into nitrogen-fixation rates requires appropriate assumptions and calibration.

Gene-expression analysis, protein detection, microscopy and isotope-based methods can provide complementary information about which organisms are present, whether nitrogenase is being produced and whether nitrogen fixation is occurring. Each method addresses a different question, and combining several approaches can provide a more reliable interpretation than relying on one measurement. This distinction is especially important in environmental microbiology, where microbial communities contain many organisms whose metabolic activities cannot be inferred directly from their genetic sequences.

Can Biological Nitrogen Fixation Reduce Fertilizer Dependence?

Biological nitrogen fixation already contributes to nitrogen supply in agricultural and natural ecosystems. Legume–rhizobium partnerships are particularly important because they can reduce the requirement for external nitrogen inputs under suitable conditions. However, the amount of nitrogen supplied varies according to plant species, bacterial strains, soil conditions and agricultural management. Effective nitrogen fixation also depends on adequate plant growth and a functioning symbiotic relationship.

Researchers are investigating ways to improve beneficial plant–microbe interactions and better understand the genetic and physiological requirements for nitrogen fixation. Studies of NodD signalling, nitrogenase regulation and microbial metabolism may eventually inform approaches to improving symbiotic efficiency. Nevertheless, establishing nitrogen fixation in crops that do not naturally form these partnerships is considerably more complicated than introducing a single gene or enzyme. Functional nitrogenase requires coordinated expression of multiple proteins, assembly of metal-containing cofactors, substantial energy and protection from oxygen damage.

Current research supports continued investigation of biological approaches to nitrogen management, but it does not establish that common cereal crops can already replace nitrogen fertilizer through engineered nitrogen fixation. Any practical improvement must be demonstrated through reliable measurements of plant growth, nitrogen acquisition and agricultural performance.

Conclusion

Biological nitrogen fixation allows certain microorganisms to convert atmospheric nitrogen into compounds that support life. The process depends on nitrogenase, a complex enzyme system requiring energy, electrons and suitable physiological conditions. Nitrogen-fixing bacteria and archaea occur in diverse environments, including soils, plant root nodules, marine waters, anaerobic sediments and high-temperature habitats.

Recent research has revealed additional mechanisms of nitrogenase regulation, new evidence for nitrogen fixation within marine particles and unusual evolutionary relationships between nitrogen-fixing microorganisms and their hosts. The discovery of the nitroplast is particularly significant because it provides evidence of a nitrogen-fixing endosymbiont becoming integrated into a eukaryotic cell. Together, these findings broaden our understanding of microbial nitrogen metabolism while emphasizing the need to distinguish the presence of nitrogenase genes from actual nitrogen-fixation activity.

Understanding these processes is important not only for microbial physiology and environmental microbiology but also for evaluating future applications in agriculture and biotechnology. Biological nitrogen fixation remains a fundamental example of how microorganisms influence nutrient availability and support life across ecosystems.

References

1. Coale, T. H., Loconte, V., Turk-Kubo, K. A., Vanslembrouck, B., Mak, W. K. E., Cheung, S., Ekman, A., Chen, J.-H., Hagino, K., Takano, Y., Nishimura, T., Adachi, M., Le Gros, M., Larabell, C., & Zehr, J. P. (2024). Nitrogen-fixing organelle in a marine alga. Science, 384(6692), 217–222. https://doi.org/10.1126/science.adk1075
2. Coët, A., Grosso, O., Brouilly, N., Ronceray, P., & Benavides, M. (2026). Micro-scale organization of non-cyanobacterial diazotrophs drives nitrogen fixation in marine particles. Communications Biology. Advance online publication. https://doi.org/10.1038/s42003-026-10971-w
3. Dixon, R., & Kahn, D. (2004). Genetic regulation of biological nitrogen fixation. Nature Reviews Microbiology, 2(8), 621–631. https://doi.org/10.1038/nrmicro954
4. Kashyap, R., Deere, T. M., Dhamad, A., Chanderban, M., Tokmina-Lukaszewska, M., Bothner, B., Lessner, D. J., & Antony, E. (2026). Cryo-EM structure of a methanogen nitrogenase–PII protein supercomplex. Nature. Advance online publication. https://doi.org/10.1038/s41586-026-11116-z
5. Kuypers, M. M. M., Marchant, H. K., & Kartal, B. (2018). The microbial nitrogen-cycling network. Nature Reviews Microbiology, 16(5), 263–276. https://doi.org/10.1038/nrmicro.2018.9
6. Maslać, N., Törer, M. R., Bolte, P., & Wagner, T. (2026). Molecular basis of N₂ fixation in a hyperthermophilic archaeon. Nature Communications, 17, Article 9602. https://doi.org/10.1038/s41467-026-77173-0
7. Reeder, C. F., von Friesen, L. W., Lemke, J., Spilling, K., & Farnelid, H. (2026). Composition of non-cyanobacterial diazotrophs in suspended and sinking particles is driven by productivity phase. Microbiology Spectrum, 14(9), e01386-26. https://doi.org/10.1128/spectrum.01386-26
8. Ruan, Y., Dong, S., Jiang, S., Wang, Y., Wu, X., Zhuang, Y., Wu, W., East, A. K., Xu, P., Poole, P. S., Zhang, Y., & Murray, J. D. (2026). The molecular basis of the binding and specific activation of rhizobial NodD by flavonoids. Science, 391(6781), 184–189. https://doi.org/10.1126/science.aec3061
9. Turk-Kubo, K., Mahaffey, C., Hopkins, J., Hawley, R., Norman, L., Wrightson, L., Fisher, B., Rynders, S., Neil, C., Ilyas, M., & Lohan, M. C. (2026). High nitrogen fixation and Braarudosphaera presence in the North Sea. Limnology and Oceanography, 71(3), e70331. https://doi.org/10.1002/lno.70331
10. Underwood, T. J., & Poole, P. S. (2026). Resource allocation to pea plant nodules impacted by nitrogen fixation potential of infecting rhizobia. The ISME Journal, 20(1), wrag097. https://doi.org/10.1093/ismejo/wrag097