Nutrient Cycles: Nitrogen

This lesson covers the nitrogen cycle, including nitrogen fixation, nitrification, denitrification, ammonification, and the role of microorganisms, as required by the Edexcel A-Level Biology specification (9BI0), Topic 10 -- Ecosystems.

Why Is Nitrogen Important?

Nitrogen is an essential element for life. It is a component of:

Amino acids (and therefore proteins)
Nucleotides (and therefore DNA and RNA)
ATP (adenosine triphosphate)
Chlorophyll

Although the atmosphere is approximately 78% nitrogen gas (N2), most organisms cannot use N2 directly because the triple covalent bond (N≡N) is extremely strong and unreactive (bond energy ~944 kJ mol^{-1}). Nitrogen must be converted into a usable form (NH4+ or NO3-) before it can be incorporated into biological molecules. This conversion process is called nitrogen fixation and is carried out only by certain specialised microorganisms.

Overview of the Nitrogen Cycle

flowchart TB
    A["N2 in\natmosphere\n(78%)"] -->|"Nitrogen fixation\n(Rhizobium, Azotobacter,\nlightning, Haber process)"| B["NH4+\n(ammonium ions\nin soil)"]
    B -->|"Nitrification Step 1\n(Nitrosomonas)\nAEROBIC"| C["NO2-\n(nitrite ions)"]
    C -->|"Nitrification Step 2\n(Nitrobacter)\nAEROBIC"| D["NO3-\n(nitrate ions\nin soil)"]
    D -->|"Absorption by\nplant roots\n(active transport)"| E["Nitrogen in\nplant proteins\nand nucleic acids"]
    E -->|"Feeding"| F["Nitrogen in\nconsumer proteins"]
    E -->|"Death /\nexcretion"| G["Dead organic\nmatter / urea"]
    F -->|"Death /\nexcretion"| G
    G -->|"Ammonification\n(decomposers)"| B
    D -->|"Denitrification\n(Pseudomonas)\nANAEROBIC"| A

Key Processes in the Nitrogen Cycle

1. Nitrogen Fixation

Nitrogen fixation is the conversion of atmospheric N2 into ammonia (NH3) or ammonium ions (NH4+) -- a form that can be used by living organisms.

There are three mechanisms:

Mechanism	Description	Estimated Contribution
Biological fixation (mutualistic)	Rhizobium bacteria living in root nodules of leguminous plants (peas, beans, clover) fix N2 using the enzyme nitrogenase. The plant provides the bacteria with glucose; the bacteria provide the plant with NH4+ -- a mutualistic relationship	~100 million tonnes N/year globally
Biological fixation (free-living)	Free-living soil bacteria such as Azotobacter and cyanobacteria fix N2 independently	~30 million tonnes N/year
Non-biological fixation	Lightning provides enough energy to combine N2 with O2, forming nitrogen oxides (NOx), which dissolve in rain. The Haber process (industrial) fixes N2 into NH3 for fertiliser production	Lightning: ~5 million tonnes; Haber process: ~150 million tonnes

Exam Tip: The enzyme nitrogenase is inhibited by oxygen, which is why Rhizobium root nodules contain leghaemoglobin -- a pink-coloured protein that binds oxygen and maintains anaerobic conditions for the nitrogenase enzyme. If you are asked to explain how Rhizobium fixes nitrogen, mention the enzyme nitrogenase, the anaerobic requirement, and leghaemoglobin.

2. Ammonification (Decomposition)

Ammonification is the conversion of organic nitrogen (in proteins, nucleic acids, urea) into ammonium ions (NH4+) by decomposers.

When organisms die, or produce waste (urea, uric acid, faeces), saprophytic bacteria and fungi break down the nitrogen-containing organic molecules
Proteases and other enzymes hydrolyse proteins into amino acids
Deamination of amino acids releases NH3 / NH4+
This returns nitrogen to the soil in a form that can be used by nitrifying bacteria

The process of deamination in the liver of animals produces urea:

$\text{Amino acid} \xrightarrow{\text{deaminase}} \text{Keto acid} + \text{NH}_3 \xrightarrow{\text{ornithine cycle}} \text{Urea (CO(NH}_2\text{)}_2\text{)}$

Urea is excreted in urine and rapidly broken down by soil bacteria (urease enzyme) to release NH4+.

3. Nitrification

Nitrification is the oxidation of ammonium ions to nitrite and then to nitrate, carried out by chemoautotrophic bacteria in the soil:

Step	Bacteria	Reaction	Conditions Required
Step 1	Nitrosomonas	NH4+ --> NO2- (nitrite)	Aerobic; warm; neutral pH
Step 2	Nitrobacter	NO2- --> NO3- (nitrate)	Aerobic; warm; neutral pH

These bacteria are chemoautotrophs -- they obtain energy from the oxidation of inorganic molecules (NH4+ or NO2-) rather than from sunlight or organic food. This links to the concept of autotrophic nutrition: not all autotrophs are photosynthetic.

Nitrate (NO3-) is the form of nitrogen most readily absorbed by plant roots via active transport.

Exam Tip: Nitrification requires aerobic conditions. Waterlogged or compacted soils with poor aeration have slow nitrification rates. This is why farmers plough and aerate the soil -- it promotes nitrification and makes more NO3- available for crop uptake.

4. Assimilation

Assimilation is the uptake of inorganic nitrogen (mainly NO3- and some NH4+) by plants and its incorporation into organic molecules:

Plant roots absorb NO3- by active transport (against the concentration gradient, requiring ATP)
NO3- is reduced to NH4+ inside the plant (using nitrate reductase)
NH4+ is combined with organic acids (from respiration, e.g. alpha-ketoglutarate) to form amino acids (via transamination)
Amino acids are used to synthesise proteins, nucleic acids, chlorophyll, and other nitrogen-containing molecules
When animals eat plants, they assimilate the nitrogen by digesting and absorbing amino acids

5. Denitrification

Denitrification is the conversion of nitrate ions (NO3-) back into atmospheric nitrogen gas (N2), carried out by denitrifying bacteria such as Pseudomonas:

$\text{NO}_3^- \rightarrow \text{NO}_2^- \rightarrow \text{N}_2\text{O} \rightarrow \text{N}_2$

Denitrification occurs in anaerobic (waterlogged) conditions
These bacteria use NO3- as an alternative electron acceptor to O2 in their respiration (anaerobic respiration)
Denitrification reduces the fertility of soil because it removes usable nitrogen
This is why waterlogged soils are less fertile

Summary of Nitrogen Cycle Bacteria

Process	Bacteria	Conditions	Conversion	Type of Nutrition
Nitrogen fixation	Rhizobium (mutualistic), Azotobacter (free-living)	Anaerobic (for nitrogenase)	N2 --> NH4+	Chemoautotroph (Azotobacter); mutualist (Rhizobium)
Nitrification (step 1)	Nitrosomonas	Aerobic	NH4+ --> NO2-	Chemoautotroph
Nitrification (step 2)	Nitrobacter	Aerobic	NO2- --> NO3-	Chemoautotroph
Denitrification	Pseudomonas	Anaerobic	NO3- --> N2	Heterotroph (uses NO3- as electron acceptor)
Ammonification	Various saprophytic bacteria and fungi	Aerobic or anaerobic	Organic N --> NH4+	Saprotroph

Common Misconception: Students often confuse nitrification and nitrogen fixation. Nitrogen fixation converts atmospheric N2 into NH4+ (bringing new nitrogen into the soil). Nitrification converts NH4+ into NO3- (changing the form of nitrogen already in the soil). These are completely different processes carried out by different bacteria.

Agricultural Implications

Practice	Effect on Nitrogen Cycle	Explanation
Adding fertilisers (e.g. ammonium nitrate)	Increases available NH4+ and NO3- for plant uptake	Risk of eutrophication if excess runs off into water (links to Lesson 9)
Ploughing / aerating soil	Promotes nitrification; reduces denitrification	Increases O2 in soil, favouring aerobic nitrifying bacteria
Crop rotation with legumes	Enriches soil nitrogen naturally	Legumes with Rhizobium fix atmospheric N2; ploughing the crop in releases nitrogen
Waterlogging soil	Promotes denitrification; reduces nitrification	Anaerobic conditions favour denitrifying bacteria
Liming (adding calcium carbonate)	Improves soil pH for nitrifying bacteria	Nitrifying bacteria work best at neutral pH

Worked Example 1: Crop Rotation

Question: Explain why a farmer might include a leguminous crop such as clover in a crop rotation.

Answer:

Clover is a legume that has Rhizobium bacteria in root nodules on its roots. These bacteria contain the enzyme nitrogenase, which catalyses nitrogen fixation -- the conversion of atmospheric N2 into NH4+ (ammonium ions). The ammonium ions are used by the clover to make amino acids and proteins. When the clover is ploughed back into the soil (green manure), decomposers break down the nitrogen-rich plant material, releasing NH4+ through ammonification. Nitrifying bacteria (Nitrosomonas and Nitrobacter) then convert NH4+ to NO3- (nitrification), which is readily absorbed by the roots of the next crop. This natural process reduces the need for artificial fertilisers, saving money and reducing the risk of eutrophication.

Worked Example 2: Interpreting Experimental Data

Question: A student measures the concentration of nitrate ions in soil samples taken from two fields: Field A is well-drained loam soil; Field B is waterlogged clay soil. Field A has a nitrate concentration of 45 mg/kg; Field B has 12 mg/kg. Both fields received the same fertiliser application. Explain the difference.

Answer:

Field A (well-drained) has a higher nitrate concentration because the aerobic conditions promote nitrification (the conversion of NH4+ to NO3- by Nitrosomonas and Nitrobacter). These chemoautotrophic bacteria require oxygen for their metabolic reactions.

Field B (waterlogged) has a lower nitrate concentration for two reasons: (1) the anaerobic conditions inhibit nitrification (less NH4+ is being converted to NO3-), and (2) the anaerobic conditions promote denitrification by Pseudomonas bacteria, which convert existing NO3- back to N2 gas, removing it from the soil. The combination of reduced nitrification and increased denitrification explains why waterlogged soil has much lower nitrate availability and is therefore less fertile.

Links to Other Specification Areas

The nitrogen cycle connects to several other topics:

Biological molecules (Topic 1): Nitrogen is a component of amino acids, proteins, nucleic acids, and ATP
Enzymes (Topic 1): Nitrogenase, urease, nitrate reductase -- all enzymes involved in nitrogen cycling
Transport in plants (Topic 4): Active transport of NO3- ions by root hair cells
Respiration (Topic 5): Denitrifying bacteria use NO3- as an alternative electron acceptor in anaerobic respiration
Gene expression (Topic 6): Nitrogen is needed for DNA and RNA synthesis
Ecology (Topic 10): Eutrophication (Lesson 9) results from excess nitrogen and phosphorus entering waterways

Summary

Nitrogen is essential for amino acids, proteins, nucleic acids, and ATP.
Atmospheric N2 must be fixed into NH4+ (by Rhizobium, Azotobacter, or lightning) before organisms can use it.
Ammonification converts organic nitrogen from dead matter into NH4+.
Nitrification (Nitrosomonas then Nitrobacter) converts NH4+ to NO2- to NO3- in aerobic conditions.
Denitrification (Pseudomonas) converts NO3- back to N2 in anaerobic conditions, reducing soil fertility.
Plants absorb NO3- by active transport and assimilate nitrogen into organic molecules.
Agricultural practices (fertilisers, crop rotation, ploughing, liming) directly affect the nitrogen cycle.

A-Level Deep Dive: The Nitrogen Cycle

Spec mapping

The Edexcel 9BI0 specification places the nitrogen cycle in Topic 5: On the Wild Side — Photosynthesis, Energy and Ecosystems, on Paper 2 (Energy, Exercise and Coordination). This lesson is the second matter-cycling counterpart to the energy-flow lesson (lesson 2), running in parallel with the carbon cycle (lesson 3) but with a fundamentally different bottleneck: where carbon-fixation chemistry (photosynthesis) is widespread, nitrogen fixation is restricted to a narrow set of prokaryotes carrying the nitrogenase enzyme — and this restriction is what makes nitrogen the limiting nutrient in most natural ecosystems. Statements concern: the role of nitrogen fixation by free-living soil bacteria (e.g. Azotobacter), symbiotic root-nodule bacteria (Rhizobium in legumes) and abiotic processes (lightning, industrial Haber–Bosch) in converting atmospheric $\text{N}_2$ to biologically available $\text{NH}_4^+$ ; the role of nitrification by chemoautotrophic Nitrosomonas ( $\text{NH}_4^+ \to \text{NO}_2^-$ ) and Nitrobacter ( $\text{NO}_2^- \to \text{NO}_3^-$ ) under aerobic soil conditions; the role of assimilation in incorporating $\text{NO}_3^-$ into amino acids and nucleic acids in producers and (via trophic transfer) consumers; the role of ammonification by saprotrophic decomposers in returning organic nitrogen to the soil as $\text{NH}_4^+$ ; and the role of denitrification by anaerobic bacteria (e.g. Pseudomonas) in returning $\text{NO}_3^-$ to the atmosphere as $\text{N}_2$ under waterlogged conditions (refer to the official Pearson Edexcel 9BI0 specification document for exact wording). The anthropogenic perturbation runs through the Haber–Bosch industrial fixation route ( $\text{N}_2 + 3\text{H}_2 \to 2\text{NH}_3$ , ~450 °C, ~200 atm, iron catalyst), which now contributes roughly half of all biologically available nitrogen worldwide and underpins modern food security. Synoptic links radiate to Topic 1 — Biological Molecules (amino acids, proteins, nucleic acids and ATP all depend on supplied N), to Topic 7 — Run for Your Life (legume root nodules and nitrogen-fixing symbiosis as an exchange-and-transport case study), to lesson 3 — Carbon Cycle (the parallel matter cycle with a different bottleneck), to lesson 2 — Energy Transfer (because protein synthesis at every trophic level requires fixed N), and to lesson 9 — Agriculture and Pollution (synthetic-fertiliser runoff causing eutrophication; $\text{N}_2\text{O}$ as a potent greenhouse-gas feedback into the climate system).

Worked example with full mark scheme

Question (8 marks):

A field of clover (a leguminous crop) is established on previously bare arable land. After one growing season, soil nitrogen analysis gives the following: total fixed N added to soil $= 180\,\text{kg ha}^{-1}\text{ yr}^{-1}$ from biological fixation in root nodules; soil $\text{NH}_4^+$ at end of season $= 12\,\text{kg ha}^{-1}$ ; soil $\text{NO}_3^-$ at end of season $= 95\,\text{kg ha}^{-1}$ ; loss to denitrification (waterlogged patches) $= 25\,\text{kg ha}^{-1}\text{ yr}^{-1}$ .

(a) Identify the bacterium responsible for nitrogen fixation in the clover root nodules and state why this fixation is energetically expensive. (2)

(b) Calculate the percentage of fixed N that has been converted to $\text{NO}_3^-$ by the end of the growing season, and identify the two bacterial genera that perform this conversion. (3)

(c) Explain three reasons why the farmer might subsequently plough the clover into the soil rather than harvest it as fodder. (3)

Solution with mark scheme:

(a) M1 (AO1.1) — Rhizobium (specifically Rhizobium leguminosarum and related genera) lives mutualistically in the root nodules of legumes; it carries the enzyme nitrogenase, which catalyses $\text{N}_2 + 8\text{H}^+ + 8\text{e}^- + 16\text{ATP} \to 2\text{NH}_3 + \text{H}_2 + 16\text{ADP} + 16\text{P}_i$ . A1 (AO1.2) — fixation is energetically expensive because the atmospheric $\text{N}_2$ molecule has a triple covalent bond (bond enthalpy ~944 kJ mol⁻¹) that requires very high activation energy to break; nitrogenase achieves this at ambient temperature only by hydrolysing 16 ATP per $\text{N}_2$ fixed, which is supplied by the legume host (the legume provides photosynthate; the bacterium provides $\text{NH}_4^+$ ).

(b) M1 (AO2.1) — percentage converted to $\text{NO}_3^-$ = (95 / 180) × 100 = 52.8% (accept 52–53%). M1 (AO1.2) — the two-step nitrification pathway is performed by Nitrosomonas ( $\text{NH}_4^+ \to \text{NO}_2^-$ ) and Nitrobacter ( $\text{NO}_2^- \to \text{NO}_3^-$ ). A1 (AO1.2) — both are chemoautotrophs that obtain energy by oxidising inorganic nitrogen substrates and require aerobic soil conditions; the residual 12 kg ha⁻¹ as $\text{NH}_4^+$ represents incomplete nitrification (likely transient pool awaiting oxidation).

(c) M1 (AO1.2) — ploughing the clover in (green manure) supplies dead organic matter that saprotrophic decomposers (bacteria, fungi) break down via ammonification, releasing further $\text{NH}_4^+$ for nitrification and uptake by the next crop. M1 (AO3.1a) — this substitutes for synthetic fertiliser, reducing input cost and reducing the risk of nitrate runoff into watercourses (eutrophication risk reduced because release is gradual rather than pulsed). M1 (AO3.2a) — crop rotation with legumes maintains soil fertility long-term: ~180 kg ha⁻¹ yr⁻¹ of biological fixation roughly matches the N demand of a typical cereal crop, so a legume-cereal rotation can sustain yields without continuous Haber–Bosch fertiliser inputs and reduces $\text{N}_2\text{O}$ greenhouse-gas emissions associated with synthetic-N application.

Total: 8 marks.

Specimen question modelled on the Edexcel 9BI0 paper format

Question (6 marks): Explain how the nitrogen cycle makes atmospheric nitrogen available to producers, and evaluate why the industrial Haber–Bosch process now constitutes a perturbation of qualitatively different scale to natural fixation.

Mark scheme decomposition by AO: