Nitrogen fixation is the conversion of atmospheric N into ammonia, usable by plants. Despite 78% of air being N, plants cannot use it directly. Microbes do this job. CBSE Class 11 and NEET test this in Mineral Nutrition and Microbes chapters.
Core Concepts
Why nitrogen fixation matters
N has a triple bond and is chemically inert. Plants need reduced nitrogen (NH or NO) for amino acids and nucleic acids. Fixation bridges the gap between atmospheric N and biological nitrogen.
The NN triple bond has a bond energy of 945 kJ/mol — one of the strongest bonds in nature. Breaking it requires enormous energy. Yet biological systems do it at room temperature and atmospheric pressure, while the Haber process needs 450°C and 200 atm.
Nitrogen budget: About 78% of the atmosphere is N, but this form is unavailable to most organisms. Only certain prokaryotes possess the enzyme nitrogenase that can break the triple bond. Without these organisms, life as we know it would not exist — nitrogen is the fourth most abundant element in living organisms (after C, H, O).
Biological nitrogen fixation
Done by nitrogenase enzyme in prokaryotes. Free-living fixers — Azotobacter, Clostridium, cyanobacteria like Nostoc and Anabaena. Symbiotic — Rhizobium in legume nodules, Frankia in non-legume trees.
The nitrogenase reaction:
Key facts about nitrogenase:
- Requires 16 ATP per N molecule fixed — extremely energy-expensive
- Contains iron (Fe) and molybdenum (Mo) at its active site
- Irreversibly inactivated by oxygen — must be protected from O
- Produces H as an obligatory by-product (wastes some energy)
Free-living nitrogen fixers:
| Organism | Type | Aerobic/Anaerobic | Habitat |
|---|---|---|---|
| Azotobacter | Bacteria | Aerobic | Soil |
| Clostridium | Bacteria | Anaerobic | Soil |
| Rhodospirillum | Bacteria | Anaerobic (photosynthetic) | Aquatic |
| Nostoc | Cyanobacteria | Aerobic (heterocysts) | Soil/water |
| Anabaena | Cyanobacteria | Aerobic (heterocysts) | Soil/water/fern symbiosis |
How cyanobacteria protect nitrogenase: Nostoc and Anabaena develop specialised thick-walled cells called heterocysts. These cells lack photosystem II (which produces O) and have thick walls that restrict O entry. Nitrogenase operates safely inside heterocysts while normal cells perform photosynthesis.
Rhizobium-legume symbiosis
Bacteria enter root hairs, form infection thread, reach cortex, trigger nodule formation. Inside the nodule, leghaemoglobin keeps oxygen low (nitrogenase is oxygen-sensitive). Bacteria fix N to NH and share with the plant; plant gives carbohydrates in return.
Step-by-step process:
Rhizobium recognises specific legume roots through chemical signals (flavonoids from plant, Nod factors from bacteria). Each Rhizobium species pairs with specific legumes — R. leguminosarum with peas, R. trifolii with clover.
Bacteria enter through curled root hair → form an infection thread (tubular structure) → travel through root cortex cells.
Bacteria released from infection thread into cortex cells → cortex cells divide rapidly → form the nodule. Bacteria inside cells are called bacteroids — they lose their cell wall and become the nitrogen-fixing form.
Bacteroids express nitrogenase → fix N to NH → NH is converted to amino acids (glutamine) → shared with the plant. Plant provides sucrose (energy source) and leghaemoglobin → both partners benefit.
Leghaemoglobin: A pink-coloured, iron-containing protein found in legume root nodules. It binds O and transports it at a controlled rate — enough for bacteroid respiration (ATP generation) but not enough to inactivate nitrogenase. The globin part is coded by the plant genome; the haem part by the bacterium.
NEET frequently asks: “Who synthesises leghaemoglobin?” The answer is both — the plant makes the globin protein, the bacterium makes the haem group. Neither can make the complete molecule alone.
Nitrogen cycle
Fixation (N to NH) → nitrification (NH to NO to NO) by Nitrosomonas and Nitrobacter → assimilation (plants take NO and build proteins) → ammonification (decomposers return N from dead matter) → denitrification (some bacteria return N to atmosphere).
Detailed nitrogen cycle:
| Process | Conversion | Organisms | Significance |
|---|---|---|---|
| Nitrogen fixation | N → NH | Rhizobium, Azotobacter, Nostoc | Makes N available to life |
| Nitrification | NH → NO → NO | Nitrosomonas, Nitrobacter | Plants prefer NO for uptake |
| Assimilation | NO → organic N | Plants, microbes | Build amino acids, nucleotides |
| Ammonification | Organic N → NH | Decomposers (bacteria, fungi) | Return N from dead matter |
| Denitrification | NO → N | Pseudomonas, Thiobacillus | Return N to atmosphere |
Nitrification is a two-step process:
- (ammonia to nitrite)
- (nitrite to nitrate)
Both organisms are chemoautotrophs — they derive energy from these oxidation reactions and use CO as their carbon source.
Denitrification is a problem for agriculture because it removes usable nitrogen from soil and returns it to the atmosphere. It is promoted by waterlogged, anaerobic conditions — this is why flooded rice paddies can lose significant nitrogen.
Industrial fixation
Haber process combines N and H at high temperature and pressure over an iron catalyst to make ammonia. This is the basis of chemical fertilizers and consumes about 1% of global energy.
Comparison of biological and industrial fixation:
| Feature | Biological (Rhizobium) | Industrial (Haber) |
|---|---|---|
| Temperature | 25-30°C (ambient) | 450°C |
| Pressure | 1 atm | 200 atm |
| Catalyst | Nitrogenase (Fe-Mo enzyme) | Fe with Mo promoter |
| Energy source | ATP from photosynthesis | Fossil fuels |
| Scale | ~140 million tonnes N/year globally | ~120 million tonnes N/year |
Both systems use iron and molybdenum — nature discovered the same catalytic metals billions of years before humans.
Worked Examples
Rhizobium in nodules fixes nitrogen, some of which remains in root debris and gets decomposed. Rotating pulses with cereals reduces the need for nitrogen fertilizer — a classic Indian farming practice.
Nitrogenase is destroyed by oxygen. The plant makes leghaemoglobin in the nodule to mop up oxygen, giving nodules a pink colour. Without it, fixation stops.
Waterlogged soil is anaerobic. Under these conditions, denitrifying bacteria (Pseudomonas) convert soil NO back to N gas, which escapes to the atmosphere. This is why flooded rice paddies need more nitrogen fertilizer than well-drained fields.
Common Mistakes
Saying all bacteria fix nitrogen. Only specific ones with nitrogenase can.
Writing that Rhizobium fixes nitrogen in any soil. It only does so inside legume root nodules.
Confusing nitrification and fixation. Fixation is N to NH; nitrification is NH to NO.
Saying nitrogenase works in the presence of oxygen. It is irreversibly inactivated by O. That is why heterocysts and leghaemoglobin exist — to protect nitrogenase.
Attributing leghaemoglobin entirely to the plant or entirely to the bacterium. The globin is plant-encoded, the haem is bacterium-encoded. It is a true partnership product.
Exam Weightage and Revision
NEET 2023 asked about the role of leghaemoglobin. NEET 2022 tested the difference between nitrification and denitrification. CBSE boards ask about the nitrogen cycle as a diagram-based five-mark question. This topic gives 1-2 guaranteed NEET questions.
When a question gives a scenario, identify the core mechanism first, then match it to the concepts above. Most wrong answers come from reading the scenario too quickly.
Memorise three free-living and two symbiotic nitrogen fixers with one example each. That covers PYQs.
Practice Questions
Q1. Why is nitrogenase called oxygen-labile?
Nitrogenase contains Fe-S clusters and an Fe-Mo cofactor that are irreversibly oxidised by O. Once oxidised, the enzyme permanently loses activity. This is why all nitrogen-fixing systems have evolved mechanisms to exclude or scavenge oxygen — heterocysts in cyanobacteria, leghaemoglobin in nodules, and anaerobic lifestyles in Clostridium.
Q2. What is the role of Nod factors in the Rhizobium-legume symbiosis?
Nod factors are signal molecules produced by Rhizobium in response to flavonoids secreted by legume roots. Nod factors cause root hair curling, allowing bacterial entry. They also trigger cortex cell division leading to nodule formation. The specificity of Nod factors determines which Rhizobium species can partner with which legume.
Q3. Name two chemoautotrophic bacteria involved in the nitrogen cycle.
Nitrosomonas: Oxidises NH to NO (first step of nitrification). Nitrobacter: Oxidises NO to NO (second step). Both are chemoautotrophs — they derive energy from these inorganic oxidation reactions and fix CO for their carbon needs.
Q4. Why do farmers practice crop rotation with legumes?
Legumes harbour Rhizobium in root nodules, which fix atmospheric N into ammonia. After harvest, the root residues decompose, releasing this fixed nitrogen into the soil. The subsequent cereal crop (rice, wheat) benefits from this nitrogen, reducing the need for synthetic fertilizers. This is why pulses (dal) are rotated with cereals in traditional Indian farming.
Q5. What would happen if all nitrogen-fixing organisms were eliminated?
Available nitrogen (NH, NO) in soil would gradually deplete through plant uptake and denitrification. Plants would suffer nitrogen deficiency (chlorosis, stunted growth). Without biological fixation, the only nitrogen input would be lightning (a small fraction) and industrial fertilizers. Natural ecosystems would collapse as the nitrogen cycle would be broken.
FAQs
How much nitrogen do biological fixers contribute globally? Biological nitrogen fixation contributes about 140 million tonnes of nitrogen per year — more than the Haber process (~120 million tonnes). Of this, symbiotic fixation (mainly Rhizobium) contributes about 80 million tonnes and free-living fixers contribute the rest.
Can Rhizobium fix nitrogen outside the plant? Rhizobium is a free-living soil bacterium when not in a nodule, but it does NOT fix nitrogen in free-living form. It only activates nitrogenase when inside the nodule environment, where leghaemoglobin controls oxygen levels and the plant provides energy (sucrose). The symbiosis is obligatory for fixation.
Why is molybdenum important for nitrogen fixation? Molybdenum (Mo) is a key component of the nitrogenase enzyme’s active site (Fe-Mo cofactor). Without Mo, the enzyme cannot bind and reduce N. This is why Mo deficiency in soil leads to nitrogen deficiency symptoms in plants — even if Rhizobium is present.
Nitrogen fixation is a chemistry problem that biology solved first. The Haber process copied what Rhizobium had been doing for two billion years.