What Is Bacteria’s Role in Converting Nitrogen in the Nitrogen Cycle?

Bacteria are responsible for nearly every major chemical transformation of nitrogen on Earth. From pulling inert nitrogen gas out of the atmosphere and converting it into a form plants can use, to recycling organic nitrogen back into the soil, to returning nitrogen gas to the air, bacteria orchestrate the steps that keep nitrogen moving through ecosystems. Without them, the nitrogen cycle would effectively stall, and life as we know it would run out of this essential element within a few generations of organisms. The full picture involves multiple groups of bacteria performing distinct and sometimes recently discovered reactions, each filling a niche that no other form of life can.

Nitrogen Fixation Starts the Whole Process

Atmospheric nitrogen makes up about 78% of the air, but it exists as N₂, a molecule held together by one of the strongest bonds in chemistry. Plants and animals cannot break that bond. Bacteria can. Certain bacteria produce an enzyme called nitrogenase, a complex metalloprotein built around an iron-rich cluster, that splits N₂ and combines it with hydrogen to produce ammonia. The reaction requires enormous energy, which the bacteria supply using ATP and chemical reductants delivered in a carefully coordinated sequence to the enzyme’s active site.¹ This is biological nitrogen fixation, and it is the primary natural entry point for new usable nitrogen into ecosystems.

The most familiar nitrogen-fixing bacteria are rhizobia, a diverse group of soil bacteria that form partnerships with legumes like soybeans, clover, peas, and alfalfa. In these symbioses, the plant grows specialized root structures called nodules that house the bacteria. Inside the nodules, the bacteria convert atmospheric nitrogen into ammonia, which the plant absorbs and uses for growth. In return, the plant feeds the bacteria carbon compounds.² The bacteria living inside these nodules are called bacteroids, and they operate under unusual conditions: they are growth-arrested, meaning they stop dividing, and they work in an environment with vanishingly low oxygen levels. That low-oxygen setup is critical because nitrogenase is destroyed by oxygen.³

This legume-rhizobium system is considered the most productive form of biological nitrogen fixation on land.⁴ It is also why farmers rotate crops with legumes: after a season of soybeans or clover, the soil is richer in available nitrogen for the next non-legume crop.

Free-Living Fixers That Work Alone

Rhizobia get most of the attention, but they are not the only nitrogen-fixing bacteria. Many species fix nitrogen without any plant partner at all. These free-living diazotrophs, as they are collectively called, live in soil, water, and sediments, and they are surprisingly diverse and widespread.⁵ Genera like Azotobacter, Clostridium, and Klebsiella all fix nitrogen independently. Because nitrogenase is oxygen-sensitive, many free-living fixers either live in low-oxygen environments or have evolved protective mechanisms to shield the enzyme.

The contribution of free-living fixers is harder to measure than that of legume nodules, but in natural ecosystems like grasslands and forests where legumes are not dominant, they supply a meaningful share of biologically fixed nitrogen. Research in temperate grasslands has shown that the community of free-living diazotrophs is shaped by soil fertilization and by proximity to plant roots, with fertilization history having the strongest effect on which nitrogen-fixing species are present.⁶ This matters because when synthetic fertilizers flood the soil with available nitrogen, the community of nitrogen fixers changes, and fixation rates may decline since the bacteria no longer need to do the work.

Some bacteria also fix nitrogen while living inside plant tissues without forming visible nodules. These endophytes, including species like Azoarcus in rice, colonize root interiors and contribute nitrogen in ways that are still being mapped out. Rice roots respond to endophytic colonization with metabolic shifts and signaling changes, though the interaction appears to work through pathways distinct from the well-studied legume symbiosis.⁷ This is an active area of research, because if nitrogen-fixing endophytes could be made to work efficiently in cereal crops like rice and wheat, it could reduce dependence on synthetic fertilizers.

Nitrification Converts Ammonia Into Nitrate

Once ammonia is in the soil, whether from biological fixation, decaying organic matter, or synthetic fertilizer, a different set of bacteria takes over. Nitrification is the two-step process that oxidizes ammonia first to nitrite and then to nitrate. Nitrate is the form of nitrogen that most plants absorb best, so nitrification is essential for making fixed nitrogen accessible to vegetation.

The first step, ammonia oxidation, is performed by ammonia-oxidizing bacteria (and also by some archaea). The key enzyme is ammonia monooxygenase, which catalyzes the rate-limiting reaction that kicks off the whole nitrification chain.⁸ The best-known ammonia-oxidizing bacterium is Nitrosomonas europaea, which has been a model organism for studying this reaction for decades. Its genome contains two copies of the gene encoding the active subunit of ammonia monooxygenase.⁹

The second step, nitrite oxidation to nitrate, is handled by nitrite-oxidizing bacteria like those in the genera Nitrobacter and Nitrospira. These organisms catalyze what seems like a simple chemical step, but it is a critical junction in the cycle: nitrate is both a nutrient for plants and a substrate for denitrifying bacteria that return nitrogen to the atmosphere.¹⁰

The Surprise of Comammox

For over a century, textbooks taught that nitrification always required two separate groups of bacteria working in relay: one group to oxidize ammonia to nitrite, another to oxidize nitrite to nitrate. That changed in 2015 with the discovery of comammox, short for complete ammonia oxidation. Researchers cultivated a bacterium from the genus Nitrospira, previously known only as a nitrite oxidizer, that turned out to carry the genetic machinery for both steps. A single organism could oxidize ammonia all the way to nitrate on its own.¹¹

The comammox organism Nitrospira inopinata has since become a subject of detailed study, including isotope experiments that help researchers trace its activity in environmental samples.¹² Comammox bacteria appear to thrive in low-ammonia environments where the traditional two-step relay might be less efficient. Their discovery reshaped how researchers think about nitrogen transformations in drinking water systems, soils, and aquatic sediments, and it is a good reminder that the nitrogen cycle still holds surprises.

Denitrification Returns Nitrogen to the Atmosphere

If nitrogen fixation is the entry ramp, denitrification is the exit. In oxygen-poor conditions, certain bacteria use nitrate or nitrite as substitutes for oxygen during respiration. The stepwise reduction moves nitrogen from nitrate through nitrite, then to nitric oxide, nitrous oxide, and finally to N₂ gas, which escapes back into the atmosphere. This closes the loop of the nitrogen cycle.

Denitrification is mediated by enzymes that belong to a family of molybdenum-dependent proteins, including nitrate reductases and nitrite oxidoreductases, which can operate in reverse directions depending on environmental conditions. The interplay between nitrate and nitrite reduction is a central feature of microbial nitrogen metabolism and is sensitive to the oxygen and redox state of the surrounding environment.¹³ Denitrifiers are not specialists in the same way nitrifiers are; many common soil and water bacteria, including species of Pseudomonas and Paracoccus, can switch to denitrification when oxygen runs low.

Denitrification is ecologically important because it prevents nitrate from accumulating endlessly in soils and waterways. But it also creates problems when the process stalls partway through.

Incomplete Denitrification and Nitrous Oxide

When denitrification does not run to completion, the penultimate product, nitrous oxide (N₂O), escapes into the atmosphere. Nitrous oxide is roughly 300 times more potent as a greenhouse gas than carbon dioxide and also damages the ozone layer. Incomplete denitrification is one of the main biological sources of this gas.

The problem crops up in many settings. In geothermal springs, thermophilic bacteria of the genus Thermus have been shown to carry incomplete denitrification pathways, meaning they reduce nitrate to nitrous oxide but lack the final enzyme to convert it to harmless N₂. These organisms may serve as a natural source of N₂O emissions from hot spring environments.¹⁴ In agricultural soils, the issue is even more pressing. When antibiotics or other contaminants enter the soil, they can suppress key denitrifying bacteria and inhibit the enzyme nitrous oxide reductase, the one that completes the final step. Research on the antibiotic sulfamethoxazole found that long-term exposure depleted important denitrifiers like Pseudomonas while enriching antibiotic-resistant genera, driving up N₂O accumulation.¹⁵

Even thawing permafrost has been implicated: when frozen soils containing nitrate warm up, the microbial communities that reawaken may carry out incomplete denitrification, releasing nitrous oxide.¹⁶ This is a feedback loop that climate researchers worry about: warming thaws permafrost, permafrost bacteria release N₂O, and N₂O accelerates further warming.

Anammox Bacteria Bypass the Usual Route

Discovered in the 1990s in a wastewater treatment reactor and later found in oceans, lakes, and wetlands, anammox bacteria perform one of the most unusual reactions in the nitrogen cycle. “Anammox” stands for anaerobic ammonium oxidation: these bacteria combine ammonium and nitrite directly into N₂ gas, without going through the full denitrification chain. They belong to the order Planctomycetales and have a remarkable internal structure, including a membrane-bound compartment called the anammoxosome, sometimes described as a prokaryotic organelle, where the reaction takes place.¹⁷

Anammox bacteria were identified in the Black Sea’s oxygen-free deep waters, where nutrient profiles and tracer experiments showed that ammonium diffusing upward from the anoxic zone was being consumed by these organisms below the oxygenated surface layer.¹⁸ In ocean oxygen minimum zones, anammox and conventional denitrification together account for a large share of total nitrogen loss from the water column. These zones harbor microbial communities where nearly every step of the nitrogen cycle operates simultaneously, with many individual organisms capable of performing multiple nitrogen-transforming reactions.¹⁹

Recent work has also shown that anammox bacteria can transfer electrons to solid surfaces outside the cell, using compounds like graphene oxide or electrodes as electron acceptors. In these experiments, ammonium was oxidized to N₂ through hydroxylamine as an intermediate, suggesting an alternative metabolic pathway that the bacteria can switch to depending on their surroundings.²⁰ This flexibility makes anammox bacteria especially interesting for engineered water treatment systems.

Wastewater Treatment Puts These Bacteria to Work

Nitrogen removal from sewage and industrial wastewater is one of the most direct practical applications of our understanding of the bacterial nitrogen cycle. Conventional treatment plants use engineered versions of nitrification and denitrification: aerobic tanks promote ammonia-oxidizing and nitrite-oxidizing bacteria, then anoxic tanks promote denitrifiers that convert the resulting nitrate into N₂ gas. More recently, anammox-based systems have gained traction because they skip the need for an external carbon source and use less energy.

Advances in synthetic biology and microbial ecology are pushing this further. Researchers are now working on assembling synthetic microbial communities, tailored consortia of isolated, enriched, or engineered strains chosen for their nitrogen-removal abilities. The idea is to match specific communities to specific wastewater compositions, selecting bacteria optimized for the pollutant types and nitrogen concentrations present.²¹ It is applied microbial ecology, driven by the same fundamental transformations bacteria have been performing for billions of years.

How Fertilizers Reshape the Microbial Landscape

Synthetic nitrogen fertilizers have doubled global food production, but they have also disrupted the microbial communities that naturally regulate the nitrogen cycle. A ten-year field experiment testing five different nitrogen application rates found that long-term fertilization reduced the number of bacterial species in soil and significantly shifted which types dominated. The primary mechanism was soil acidification: the more fertilizer applied, the lower the soil pH dropped, and many bacteria that thrived in the original soil were replaced by acid-tolerant species.²²

The form of the fertilizer matters too. Whether nitrogen is applied as a synthetic compound or as manure affects soil chemistry differently, which in turn alters the bacterial contribution to N₂O emissions. Even when the overall microbial community structure does not change dramatically between fertilizer types, shifts in soil chemical properties still drive differences in how much nitrous oxide bacteria produce.²³

One strategy to manage this is nitrification inhibitors: chemical compounds applied alongside fertilizer that slow the conversion of ammonia to nitrate, keeping nitrogen in the soil longer so plants have more time to absorb it. Synthetic nitrification inhibitors like DMPP (dimethylpyrazole phosphate) work by blocking ammonia-oxidizing bacteria. Research has shown that these inhibitors do not just reduce nitrification; they also stimulate the final step of denitrification, encouraging bacteria to fully reduce N₂O to harmless N₂ rather than letting it escape as a greenhouse gas.²⁴ Biological nitrification inhibitors, compounds naturally exuded by certain plant roots, are also being explored as a more sustainable alternative.²⁵

The Deep History of Nitrogen-Fixing Bacteria

Nitrogen fixation is not a recent evolutionary invention. Molecular clock analyses and phylogenetic reconstructions suggest that the ability to fix nitrogen first arose in anaerobic bacteria, likely more than 2.5 billion years ago, at a time when Earth’s atmosphere contained almost no free oxygen. Aerobic nitrogen fixation in bacteria, including the ancestors of modern cyanobacteria, appears to have evolved later, after these organisms developed defenses against oxygen damage. Archaea, which also include some nitrogen fixers, seem to have acquired the ability at roughly the same time as or slightly after aerobic bacteria.²⁶

The genes responsible for nitrogen fixation have a complex evolutionary tree of their own. The major groups of nitrogenase genes appear to have diverged during or after the breakup of the ancient supercontinent Kenorland, roughly 2.5 to 2.1 billion years ago. One hypothesis is that the continental breakup physically separated bacterial populations into isolated groups, accelerating the diversification of nitrogen-fixation pathways.²⁷ This deep history underscores how fundamental nitrogen-cycling bacteria are to the planet’s biogeochemistry: they have been doing this work for the majority of Earth’s history, long before plants, animals, or even oxygen-rich atmospheres existed.

Microplastics and Emerging Disruptions

The bacterial nitrogen cycle is not just affected by fertilizers. Microplastics, now ubiquitous in soils worldwide, are altering nitrogen transformations in ways that depend on the type of plastic. Polyethylene microplastics have been found to increase ammonia emissions by roughly 20 to 34% and also boost methane output, while polyvinyl chloride (PVC) microplastics reduce both of those emissions but through a mechanism that also interferes with nitrate concentrations and compost maturity. Both polyethylene and PVC lead to increased nitrous oxide emissions compared to uncontaminated controls.²⁸ The implication is that as microplastic pollution grows, the efficiency and balance of nitrogen cycling in agricultural soils may shift in unpredictable directions, adding yet another variable to an already complex system.

Measuring What Bacteria Actually Do in the Field

One reason our understanding of the nitrogen cycle has evolved so much in recent decades is improved methods for measuring bacterial activity in real environments. A classic technique for estimating nitrogen fixation is the acetylene reduction assay: bacteria are incubated with acetylene gas, and the nitrogenase enzyme reduces it to ethylene, which can be measured with a gas chromatograph. The amount of ethylene produced gives a proxy for nitrogen-fixing activity.²⁹ Newer molecular techniques track specific genes, like nifH for nitrogen fixation or amoA for ammonia oxidation, to identify which organisms are present and whether they are actively expressing the genes in question.³⁰ The gap between possessing a gene and actually using it is significant; just because a bacterium carries the genetic potential for nitrogen fixation or nitrification does not mean it is doing so at any given moment. Environmental conditions like oxygen level, nutrient availability, and pH all govern when these genes switch on.

These tools have been essential for discoveries like comammox and for quantifying the relative importance of anammox versus denitrification in ocean nitrogen loss. They have also revealed that the microbial nitrogen cycle is more redundant and more interconnected than older models suggested, with many organisms capable of multiple nitrogen transformations depending on conditions. The picture that emerges is not a neat linear chain but a web of overlapping reactions, with bacteria occupying every node.