Nitrogen Cycle: Steps and the Role of Microorganisms

Earth's atmosphere is 78 percent nitrogen, an almost unlimited supply floating above every field and forest. Yet nitrogen is one of the nutrients that most often limits plant growth. The reason is a paradox at the heart of biology: no plant and no animal can use nitrogen gas directly. The triple bond that holds the two nitrogen atoms together is one of the strongest in nature, and breaking it is beyond the reach of every organism except a small group of microbes.

This is why the nitrogen cycle is, more than almost any other, a microbial story. At every step where nitrogen changes form, a microbe does the work. Bacteria pull nitrogen out of the air, convert it through a series of chemical forms that plants and animals can use, and finally return it to the atmosphere as gas. Remove the microbes and the cycle stops, and with it most life on land.

This article walks through the steps of the nitrogen cycle in order, then focuses on the organisms that run each one.

What is the nitrogen cycle?

The nitrogen cycle is the movement of nitrogen between the atmosphere, living organisms, and the soil, changing chemical form at each step. Like the carbon cycle, it is a biogeochemical cycle: nitrogen passes through living things (bio), through soil and water (geo), and changes its chemical form (chemical) along the way.

Nitrogen exists in several forms during the cycle, and it helps to keep them straight from the start:

• Nitrogen gas (N₂): the atmospheric form, unusable by plants and animals.
• Ammonia and ammonium (NH₃ / NH₄⁺): the first usable form, produced by fixation and by decay.
• Nitrite (NO₂⁻): an intermediate form, produced during nitrification.
• Nitrate (NO₃⁻): the main form plants absorb from the soil.
• Organic nitrogen: nitrogen built into proteins and nucleic acids inside living things.

The cycle is the set of steps that convert nitrogen from one of these forms to the next.

Steps of the Nitrogen Cycle

The nitrogen cycle has five main steps. Four of the five are carried out entirely by microbes, and the fifth (assimilation) depends on the products microbes make.

1. Nitrogen fixation: nitrogen gas (N₂) is converted to ammonia (NH₃). This is the step that brings new usable nitrogen into the living world.
2. Nitrification: ammonia is oxidized first to nitrite (NO₂⁻), then to nitrate (NO₃⁻).
3. Assimilation: plants take up nitrate (or ammonium) and build it into proteins and nucleic acids. Animals get their nitrogen by eating plants.
4. Ammonification (mineralization): when organisms die or excrete waste, microbes break the organic nitrogen back down to ammonia.
5. Denitrification: nitrate is converted back to nitrogen gas, returning it to the atmosphere and closing the cycle.

The sections below explain each step and the microbes that carry it out.

Role of Microorganisms in the Nitrogen Cycle

The nitrogen cycle is the clearest example in all of biology of a process that microbes run from start to finish. Here is what happens at each step and which organisms do it.

1. Nitrogen fixation (bringing nitrogen into life)

Nitrogen fixation converts atmospheric nitrogen gas into ammonia, the first form living things can use. Only bacteria and archaea can do this, using an enzyme called nitrogenase. No plant, animal, or fungus can fix nitrogen on its own.

The fixers fall into three groups by how they live:

• Symbiotic fixers live inside a plant partner. The most important is Rhizobium, which forms nodules on the roots of legumes such as peas, beans, and soybeans, and fixes nitrogen in exchange for sugars from the plant.
• Free-living fixers work alone in the soil. Examples are Azotobacter (aerobic) and Clostridium (anaerobic).
• Cyanobacteria fix nitrogen in water and wet soils. In rice paddies, the water fern Azolla houses the cyanobacterium Anabaena azollae in its leaves and supplies nitrogen to the crop.

Nitrogenase has one important weakness: it is destroyed by oxygen. Nitrogen-fixing microbes protect it in different ways, for example by working inside oxygen-free tissues or specialized cells. This oxygen sensitivity is a recurring theme in how and where fixation happens.

Because fixation supplies nitrogen without chemical fertilizer, these organisms are widely used in farming as biofertilizers and are central to the role of microbes in agriculture.

2. Nitrification (turning ammonia into nitrate)

Nitrification is the oxidation of ammonia to nitrate, and it happens in two steps carried out by two groups of bacteria:

• Ammonia to nitrite: carried out by ammonia-oxidizing bacteria such as Nitrosomonas.
• Nitrite to nitrate: carried out by nitrite-oxidizing bacteria such as Nitrobacter.

These bacteria are chemolithotrophs: they get their energy from oxidizing these inorganic nitrogen compounds rather than from organic food. Nitrification needs oxygen, so it happens in well-aerated soils. The nitrate it produces is the main form of nitrogen that plants absorb.

3. Assimilation (nitrogen enters plants and animals)

Assimilation is the step where nitrogen enters living tissue. Plants take up nitrate or ammonium from the soil through their roots and build it into amino acids, proteins, and nucleic acids.

Animals cannot use soil nitrogen at all; they get their nitrogen by eating plants or other animals. This is the one main step that is not carried out by microbes, though it depends entirely on the usable nitrogen that microbes produce.

4. Ammonification (returning nitrogen from the dead)

When plants and animals die, and when animals excrete waste, their nitrogen is locked in organic molecules. Ammonifying bacteria and fungi decompose this organic nitrogen and release it back as ammonia.

This step is also called mineralization, and it is the same decomposition work microbes do in the carbon cycle, seen from the nitrogen side. The ammonia released here can be nitrified again, keeping nitrogen in circulation.

5. Denitrification (returning nitrogen to the air)

Denitrification closes the cycle. Denitrifying bacteria such as Pseudomonas convert nitrate back into nitrogen gas, which returns to the atmosphere. This step happens in oxygen-free conditions, such as waterlogged soils, where the bacteria use nitrate instead of oxygen for respiration.

Denitrification removes usable nitrogen from the soil, which is why waterlogged fields lose fertility, but it is also what keeps nitrogen from building up indefinitely and balances the nitrogen brought in by fixation.

Read together, these five steps show why the nitrogen cycle is a microbial metabolism. Microbes bring nitrogen in (fixation), convert it into the form plants use (nitrification), release it again from the dead (ammonification), and return it to the air (denitrification). Only the uptake step belongs to plants and animals.

Microbes of the nitrogen cycle

Step	What happens	Main microbes
Nitrogen fixation	N₂ to ammonia	Rhizobium (legumes), Azotobacter, Clostridium, cyanobacteria (Anabaena)
Nitrification (step 1)	Ammonia to nitrite	Nitrosomonas
Nitrification (step 2)	Nitrite to nitrate	Nitrobacter
Ammonification	Organic nitrogen to ammonia	Many soil bacteria and fungi
Denitrification	Nitrate to N₂ gas	Pseudomonas, Thiobacillus denitrificans

Why the Nitrogen Cycle Matters

Nitrogen is a building block of every protein and every strand of DNA, so no organism can grow without it. The nitrogen cycle is what keeps nitrogen moving from the vast but unusable atmospheric supply into a form life can use, and back again.

For agriculture, the cycle is the foundation of soil fertility. Nitrogen fixation is the natural route by which nitrogen enters farmland, which is why legumes and biofertilizers matter so much, and why denitrification in waterlogged soil is a problem farmers work to avoid.

Humans have altered the cycle dramatically. The Haber-Bosch process, invented in 1909, fixes nitrogen industrially into fertilizer and now adds roughly as much usable nitrogen to the planet as all natural fixation combined.

This has fed billions of people, but the excess nitrogen also runs off into rivers and seas, causing algal blooms and dead zones, and denitrification of it releases nitrous oxide, a potent greenhouse gas. Understanding the microbial cycle is what makes these problems, and their possible solutions, make sense.

How to Remember

The five steps in order: Fix, Nitrify, Assimilate, Ammonify, Denitrify. Nitrogen is Fixed from the air, Nitrified to nitrate, Assimilated into plants, Ammonified back from the dead, and Denitrified back to the air. One sentence carries the whole cycle in sequence.

Nitrosomonas comes before Nitrobacter. In nitrification, ammonia becomes nitrite first, then nitrate. Nitrosomonas makes nitrite (both have "nitroso"); Nitrobacter makes nitrate. Alphabetical order (Nitrosomonas before Nitrobacter) is also the reaction order.

Only microbes fix nitrogen, never plants. The air is full of nitrogen, but the triple bond is too strong for any plant or animal to break. Every atom of nitrogen in a protein was either fixed by a microbe or made in a fertilizer factory. Legumes only fix nitrogen because Rhizobium lives in their roots.

Fixation and denitrification are opposites at the two ends. Fixation brings nitrogen in from the air (N₂ to ammonia); denitrification sends it back (nitrate to N₂). One opens the cycle, the other closes it. In between, the nitrogen is passed along in usable forms.

Key exam facts

Fact	Answer to remember
What kind of cycle it is	A biogeochemical cycle
Why plants cannot use N₂	The nitrogen triple bond is too strong to break without nitrogenase
Enzyme that fixes nitrogen	Nitrogenase (destroyed by oxygen)
Groups that can fix nitrogen	Only bacteria and archaea
Symbiotic nitrogen fixer	Rhizobium (in legume root nodules)
Free-living nitrogen fixers	Azotobacter (aerobic), Clostridium (anaerobic)
Ammonia to nitrite	Nitrosomonas
Nitrite to nitrate	Nitrobacter
Form plants mainly absorb	Nitrate (NO₃⁻)
Organic nitrogen back to ammonia	Ammonification (mineralization)
Nitrate back to N₂ gas	Denitrification (anaerobic, Pseudomonas)
Industrial nitrogen fixation	Haber-Bosch process (1909)

Where Students Get Confused

Nitrification versus denitrification. They sound alike but move nitrogen in opposite directions. Nitrification builds ammonia up to nitrate (and needs oxygen). Denitrification breaks nitrate back down to nitrogen gas (and needs the absence of oxygen). One adds oxygen atoms; the other removes nitrogen from the soil entirely.

Nitrogen fixation versus assimilation. Fixation converts nitrogen gas from the air into ammonia, bringing new nitrogen into the system. Assimilation is plants taking up nitrogen that is already in the soil as nitrate or ammonium. Fixation is the entry point; assimilation is uptake.

Nitrosomonas and Nitrobacter do different jobs. Nitrosomonas oxidizes ammonia to nitrite. Nitrobacter oxidizes nitrite to nitrate. They act in sequence, not interchangeably, and the names are easy to swap. Nitroso to nitrite, Nitro to nitrate.

Plants do not fix their own nitrogen. A legume appears to fix nitrogen, but the work is done by Rhizobium bacteria living in its root nodules. The plant provides the home and the sugars; the bacterium provides the nitrogenase.

Ammonification is not the same as denitrification. Both return nitrogen, but to different places. Ammonification returns organic nitrogen to the soil as ammonia, keeping it in the cycle. Denitrification returns nitrate to the atmosphere as gas, taking it out of the soil.

Denitrification is a loss, not a gain, for the soil. Students sometimes assume every step adds usable nitrogen. Denitrification does the opposite: it removes nitrate from the soil and sends it to the air, which is why it reduces soil fertility.

Recent understanding

The classical five-step cycle is the core, but microbiologists now recognize additional pathways. The most important is anammox (anaerobic ammonium oxidation), in which specialized bacteria convert ammonium and nitrite directly into nitrogen gas without going through the full nitrate route.

Anammox is now known to account for a large share of the nitrogen returned to the atmosphere from the oceans. The take-home is that the real nitrogen cycle is a network of overlapping microbial reactions, not a single tidy loop, though the five classical steps remain the right framework for learning it.

References

1. Willey JM, Sandman K, Wood D (2020). Prescott's Microbiology. 11th edn. McGraw-Hill Education.
2. Madigan MT, Bender KS, Buckley DH, Sattley WM, Stahl DA (2021). Brock Biology of Microorganisms. 16th edn. Pearson. pp. 645-672.
3. Kuypers MMM, Marchant HK, Kartal B (2018). The microbial nitrogen-cycling network. Nature Reviews Microbiology. 16(5): 263-276. https://doi.org/10.1038/nrmicro.2018.9
4. Bhuvaneshwari K, Singh PK (2015). Response of nitrogen-fixing water fern Azolla biofertilization to rice crop. 3 Biotech. 5(4): 523-529. https://doi.org/10.1007/s13205-014-0251-8