Overview — Why nitrogen metabolism matters

Living organisms are built from carbon, hydrogen, oxygen, nitrogen and other elements. Nitrogen ranks next to carbon in importance: it is a constituent of amino acids, proteins, enzymes, vitamins, alkaloids and some growth hormones, and of nitrogenous bases in nucleic acids. Life’s chemistry therefore depends on how nitrogen is fixed from the air and assimilated into organic molecules. This NIOS Biology lesson (Module 2) covers molecular nitrogen and the nitrogen cycle, abiological and biological nitrogen fixation, free-living and symbiotic fixers (especially legumes and Rhizobium), nitrate and ammonia assimilation, and amino-acid synthesis by reductive amination and transamination.

After this lesson you should be able to describe modes of nitrogen fixation; explain free-living and symbiotic fixation; describe assimilation of nitrate and ammonia; and describe amino-acid synthesis in plants. Notes follow the textbook order only.

Link forward: respiration (Krebs cycle keto acids) supplies carbon skeletons for amino acids; photosynthesis supplies reductant and ATP that drive fixation and nitrate reduction. Lesson 11 (photosynthesis) and Lesson 12 (respiration) close that loop. Treat this lesson as the “nitrogen bridge” between soil microbes, plant nutrition and protein synthesis — every later mention of amino acids or enzymes silently assumes the pathways you learn here.

Section 1: Molecular nitrogen (10.1)

Nitrogen occurs freely in the atmosphere as dinitrogen (N₂) and in combined mineral form as Chile saltpetre (sodium nitrate) — Chile in South America being a classic source of nitrate nitrogen.

Molecular nitrogen is highly stable because it is triple bonded (N≡N). Under normal atmospheric conditions it is not very reactive. Atmosphere is about 78.03% N₂ by volume. Boiling point is very low (−195.8°C), even lower than oxygen. Proteins in living organisms contain about 16% nitrogen.

Intext facts to lock: % volume of N₂; biomolecules containing N (proteins, enzymes, nucleic acids…); why N₂ is stable (triple bond); % N in protein; boiling point.

Why agriculture cares: crop yield is often limited by available soil nitrogen. Farmers add urea, ammonium salts or nitrate fertilisers — products ultimately linked to industrial Haber ammonia. Understanding biological fixation (legume rotation, green manure with Azolla) is the ecological counterpart to synthetic fertiliser. The Chile saltpetre story reminds you that nitrate also exists as a mineral deposit, but the atmosphere remains the vast reservoir of N₂ that only specialised chemistry or biology can unlock.

Nucleic acids (DNA, RNA) carry nitrogen in their bases; chlorophyll and some plant secondary metabolites also contain N. When the book lists vitamins, alkaloids and growth hormones among N-containing molecules, it is stressing that “nitrogen metabolism” is not only fertiliser science — it is central cell chemistry from gene to protein to signalling.

Section 2: Nitrogen cycle (10.1.1)

Air has ~78% N₂ but most organisms cannot use it directly. The nitrogen cycle converts atmospheric nitrogen into usable forms and returns N₂ to the air so biosphere nitrogen content stays roughly constant.

• Lightning can fix nitrogen toward ammonia/oxides.
• Nitrogen-fixing bacteria such as Rhizobium in roots of legumes (pea, rajma, beans, pulses) convert N₂ → NH₃.
• Most plants absorb nitrates from soil and reduce them to NH₃ inside cells for further metabolism.
• Dead organisms and excreta (e.g. urea) are decomposed by bacteria to NH₃ and, by other bacteria, to nitrates for plant use.
• Denitrifying bacteria convert nitrates back to nitrogen gas, closing the cycle.

The cycle is self-regulated in nature, but human activities have caused steady loss of soil nitrogen in many regions — a textbook caution about sustainability of soil fertility.

MCQ-style from intext: nitrogen fixation converts atmospheric N mainly to ammonia (option b in book). Nitrogen content of biosphere remains constant because of the nitrogen cycle. Nitrates → N₂ by denitrifying bacteria.

Trace one atom of nitrogen in a story form for long answers: lightning or Rhizobium creates combined N → plant root takes up nitrate → NR and NiR make NH₃ → glutamate and other amino acids → proteins in leaf → animal eats plant → animal protein → urea in urine → soil bacteria release NH₃ → nitrifiers form nitrate again → another plant uses it, or denitrifiers free N₂ back to air. Human harvest and erosion can short-circuit the soil pool, which is why the textbook notes “steady loss of soil nitrogen” under human pressure.

Section 3: Nitrogen fixation — definition and modes (10.2)

Nitrogen fixation is the conversion of molecular nitrogen into compounds of nitrogen, especially ammonia. It is a reductive process: fixation stops if reducing conditions fail or if oxygen is present (oxygen damages or blocks nitrogenase activity). Two broad methods: abiological and biological.

3.1 Abiological nitrogen fixation

Reduction of N₂ to ammonia (or formation of nitrogen compounds) without any living cell. Two subtypes:

• Industrial — Haber process: mixture of N₂ and H₂ passed over iron-oxide catalyst at very high temperature (~500°C) and pressure (~1000 atm) to give synthetic ammonia: N₂ + 3H₂ → 2NH₃.
• Natural: especially during electrical discharges / lightning storms, nitrogen combines with oxygen to form oxides of nitrogen, which hydrate and reach earth as nitrite and nitrate.

3.2 Biological nitrogen fixation

Chemically the same reduction idea, but carried out by a living cell using the enzyme nitrogenase. Distinguish: biological = living cell + nitrogenase; abiological = no living cell (Haber or lightning chemistry).

Section 4: Free-living and symbiotic fixation (10.3)

Only selected organisms possess nitrogenase. Fixation is mainly in bacteria and cyanobacteria. They may be free-living or symbiotic.

4.1 Free-living microbes (Table 10.1)

• Clostridium — anaerobic, non-photosynthetic bacteria.
• Klebsiella — facultative, non-photosynthetic.
• Azotobacter — aerobic, non-photosynthetic.
• Rhodospirillum — purple non-sulphur photosynthetic bacteria.
• Anabaena — cyanobacteria (photosynthetic).

4.2 Symbiotic systems (Table 10.2)

Microbe + host form symbiosis → symbiotic nitrogen fixation:

• Lichens — cyanobacteria + fungus.
• Bryophyte — cyanobacteria + Anthoceros.
• Pteridophyte — cyanobacteria + Azolla.
• Gymnosperm — cyanobacteria + Cycas.
• Angiosperm legumes — Rhizobium.
• Non-legume angiosperms (Alnus, Myrica, Purshia) — actinomycete.
• Brazilian grass Digitaria, corn — Azospirillum.

Cyanobacteria are the symbiotic partner in lichens, bryophytes, pteridophytes and gymnosperms; in legumes the partner is Rhizobium.

4.3 Mechanism of biological fixation

Requirements: (i) molecular nitrogen; (ii) strong reducing power (reduced FAD, NAD); (iii) ATP energy to transfer hydrogen from NADH₂/FADH₂ to N₂; (iv) enzyme nitrogenase; (v) a way to trap toxic ammonia (combine with organic acids → amino acids). Reductant and ATP come from photosynthesis and respiration.

Nitrogenase is a Mo–Fe protein that binds N₂ and reduces it stepwise with hydrogen: first diamide (N₂H₂), then hydrazine (N₂H₄), finally ammonia (2NH₃). Ammonia is not liberated free; fixers incorporate it into amino acids because free NH₃ is toxic.

Overall equation (textbook): N₂ + 16ATP + 8H⁺ + 8e⁻ → 2NH₃ + 16ADP + 16P_i. Molecular nitrogen is so stable that a large ATP cost is needed. Hydrogen gas evolution may accompany the process.

4.4 Legume nodules, leghaemoglobin, nodulins

In legumes, fixation occurs in specialised root nodules formed by interaction of Rhizobium and root. Biochemical steps match free-living fixation, but nodules have special protein leghaemoglobin. Neither bacterium alone nor plant alone makes it; both partners contribute — Rhizobium gene codes for heme part, host for globin moiety. Function: oxygen scavenger — lowers partial pressure of O₂ so nitrogenase can work under anaerobic conditions while the rest of the plant remains aerobic. Free-living microbes and many cyanobacterial symbioses lack leghaemoglobin; cyanobacteria can fix under aerobic conditions by other strategies.

A group of proteins called nodulins also help establish symbiosis and maintain nodule function. Host genes are involved beyond just housing bacteria.

Matching memory: Azotobacter ↔ aerobic fixer; Clostridium ↔ anaerobic; Anabaena ↔ cyanobacterium; lichens ↔ symbiotic; Cycas ↔ gymnosperm that fixes (via cyanobacteria); 16 ATP per N₂; electrons from reduced coenzymes/ferredoxin; H₂ may evolve.

Free-living vs legume systems (exam contrast): free-living fixers live independently in soil or water and release combined N to the environment after their own use; legume systems fix N inside nodules for the plant partner in exchange for photosynthate and a protected niche. Free-living cells protect nitrogenase by respiratory O₂ consumption, thick walls, or heterocysts (in some cyanobacteria); legumes invent leghaemoglobin as an O₂ buffer. Both use the same core chemistry (nitrogenase + ATP + reductant) but differ in ecology and special proteins.

Energy source note from “what you have learnt”: electrons and energy for fixation are generally linked to respiratory metabolism — pyruvic acid entering Krebs cycle provides reducing power and ATP. Photosynthetic fixers additionally use light-driven electron transport. When the book asks “major source of electrons for reduction of nitrogen,” answer reduced coenzymes (NADH₂, FADH₂) / ferredoxin — not the nitrogen molecule itself.

Section 5: Nitrate and ammonia assimilation (10.4)

Only some organisms fix N₂. All plants need nitrogen for metabolism, so non-fixers use soil nitrate and ammonia. Nitrate is the most oxidized common inorganic form; ammonium is the most reduced.

Plants absorb nitrate and reduce it to ammonia in two enzyme steps:

(1) Nitrate reductase (cytosol): NO₃⁻ + NADH + H⁺ → NO₂⁻ + NAD⁺ + H₂O. Enzyme contains FAD, cytochrome, NADPH or NADH, and molybdenum. Energy-dependent. Continuously synthesized and degraded; inducible by rising nitrate in cytosol; excess NH₄⁺ has a negative effect on synthesis; light increases nitrate reductase when nitrate is available.

(2) Nitrite reductase (chloroplast or plastid): nitrite moves from cytosol into plastid; NO₂⁻ + 3NADPH + 3H⁺ → NH₃ + 3NADP⁺. Electrons can come from NADH, NADPH, FADH₂ or reduced ferredoxin.

Ammonia must be used quickly — accumulation is toxic. Some plants and algae leach excess ammonia, which microbes can re-oxidise to nitrite and nitrate in soil or water.

Why the two-step design matters: nitrate is safe to transport and store relative to free ammonia, yet plants must fully reduce it before incorporating N into amino acids. Putting NR in the cytosol and NiR in the chloroplast couples the second, high-electron-demand step to photosynthetic reductant (ferredoxin) in green cells. In roots, plastids still house NiR using reductant from local metabolism. Light stimulation of NR when nitrate is present links daytime photosynthesis to daytime nitrate assimilation — a coordination point examiners like when asking “effect of light on nitrate reductase.”

Negative feedback: when ammonium builds up, synthesis of nitrate reductase falls, preventing unnecessary reduction of more nitrate to ammonia. Positive signal: more nitrate induces more NR protein. Together these controls keep the reduction pipeline matched to need.

Fixation vs assimilation: fixation creates combined nitrogen (mainly NH₃) from N₂; assimilation is uptake and metabolic use of already combined forms (NO₃⁻, NH₄⁺) into organic molecules. Do not use the terms interchangeably in long answers.

Section 6: Amino acid synthesis (10.5)

Ammonium from fixation or nitrate reduction is the major inorganic source for amino acids — building blocks of proteins and enzymes. A typical amino acid has an amino group (−NH₂) and a carboxyl group (−COOH) on a carbon with side chain R.

NH₄⁺ supplies the amino group; the carboxyl-bearing carbon skeleton comes from plant organic acids (often from respiration).

6.1 Reductive amination

Ammonia combines with a keto acid. Most important keto acid: α-ketoglutaric acid from Krebs cycle. Enzyme glutamate dehydrogenase: α-ketoglutarate + NH₃ → glutamic acid. Aspartic acid forms similarly from oxaloacetic acid. Reductive amination is the major “port of entry” of ammonia into plant metabolism — glutamic acid first, then other amino acids.

6.2 Transamination

Transfer of an amino group from an already made amino acid to a keto acid, catalysed by transaminases. Example: α-ketoglutaric acid + aspartic acid ⇌ glutamic acid + oxaloacetic acid. This multiplies the variety of amino acids without needing free NH₃ for every new product.

Difference: reductive amination uses free ammonia + keto acid (dehydrogenases); transamination transfers −NH₂ between amino acid and keto acid (transaminases) and does not itself bring new inorganic N into the system — it rearranges organic N.

Write the amino-acid structure once in answers: central carbon with H, R, COOH and NH₂. Name glutamic acid as the product of α-ketoglutarate reductive amination — high-frequency one-marker. Aspartate from oxaloacetate is the parallel example. Transamination example in the book uses α-ketoglutarate + aspartate ⇌ glutamate + oxaloacetate — learn that pair so you can reverse the reaction in your head.

Connection to later lessons: Krebs cycle intermediates are not only for respiration energy; they are carbon skeletons for N assimilation. Without photosynthesis supplying sugar to roots and nodules, symbiotic fixation slows — another reason legumes need light and healthy shoots.

Section 7: Exam quick hits and checklist

From “What you have learnt” and terminal exercises:

• N in amino acids, proteins, enzymes, vitamins, alkaloids, nucleic acids, pigments, hormones.
• N₂ triple-bonded and stable; fixation = reduction to ammonia; Haber and lightning are abiological; nitrogenase is biological.
• Free-living vs symbiotic tables; cyanobacteria vs Rhizobium partners; leghaemoglobin scavenges O₂; nodulins support nodules.
• 16 ATP, stepwise reduction, possible H₂ evolution; electrons from reduced coenzymes/ferredoxin/pyruvate via respiration.
• Nitrate most oxidized, ammonium most reduced; NR in cytosol; NiR in chloroplast; NR inducible.
• Reductive amination (GDH, glutamate) vs transamination (transaminases).

Define nitrogen fixation; name forms fixed in lightning (oxides/nitrate/nitrite path); one aerobic (Azotobacter) and one anaerobic (Clostridium) fixer; amino acid from α-KG + NH₃ = glutamic acid; requirements for biological fixation; function of leghaemoglobin vs human hemoglobin (O₂ transport in blood vs O₂ scavenging for anaerobic nitrogenase); difference fixation vs assimilation; free-living vs legume systems.

One-line intext keys: 78.03% N₂; proteins/enzymes as N biomolecules; triple bond stability; 16% N in protein; −195.8°C; fixation → ammonia; cycle keeps N constant; denitrifiers; Haber; oxygen prevents fixation; Cycas; 16 ATP; nodulins; NH₄⁺ most reduced; nitrate most oxidized; cytosol for NR; chloroplast for NiR; reductive amination + transamination; transaminases; ammonia source for reductive amination; α-ketoglutaric acid for glutamate.

Use the Formula Sheet tab for lockable summaries; drill the ten MCQs and twenty flashcards. This chapter is denser on enzymes and equations than morphology chapters — prioritise definitions, enzyme names/sites, and the 16-ATP equation.

Section 8: Worked revision — terminal-style outlines

Define nitrogen fixation. Conversion of molecular nitrogen into nitrogen compounds, especially ammonia, by a reductive process (abiological or biological).

Requirements for biological fixation. N₂ substrate; strong reductant (NADH₂/FADH₂/ferredoxin); ATP; nitrogenase (Mo–Fe); organic acids to trap NH₃ as amino acids; anaerobic or low-O₂ microenvironment.

Brief abiological process. Haber: N₂ + 3H₂ at high T/P over iron catalyst → NH₃. Natural: lightning N + O → oxides → hydrated nitrites/nitrates in rain to soil.

Biological steps. Nitrogenase binds N₂; successive addition of 2e⁻/2H⁺ yields diamide, hydrazine, then two NH₃; 16 ATP hydrolysed per N₂; NH₃ rapidly aminated onto keto acids; H₂ may evolve.

Leghaemoglobin vs human hemoglobin. Both bind oxygen, but leghaemoglobin’s role in nodules is to keep free O₂ low for nitrogenase, not to transport O₂ to tissues as in blood.

NR vs NiR. NR: nitrate→nitrite, cytosol, NADH, Mo, inducible. NiR: nitrite→ammonia, plastid, multi-electron reductant/ferredoxin. Together they assimilate soil nitrate into cellular NH₃.

Reductive amination vs transamination. First adds free NH₃ to keto acid (entry of inorganic N). Second transfers organic −NH₂ between molecules (network of amino acids). Enzymes: dehydrogenases vs transaminases.

Section 9: Closed-book drill list

(1) State five biomolecules containing nitrogen. (2) Give % N₂, % N in protein, boiling point, bond type. (3) Sketch nitrogen cycle with fixation, nitrates, denitrification. (4) Haber conditions and equation. (5) Lightning products. (6) Free-living table (one aerobic, one anaerobic, one cyanobacterium). (7) Symbiotic partners for lichen, Azolla, Cycas, pea. (8) Five requirements of biological fixation. (9) Stepwise intermediates and 16 ATP equation. (10) Leghaemoglobin function and genetic origin of parts. (11) Nodulins. (12) NR and NiR sites and products. (13) Inducible nature of NR. (14) Most oxidized vs most reduced inorganic N. (15) Reductive amination equation for glutamate. (16) One transamination example. (17) Fixation vs assimilation in one sentence each. (18) Why free NH₃ is not left free in cells.

If you can answer all eighteen without notes, Lesson 10 is exam-ready. Next plant physiology chapter in the public exam set is often photosynthesis (L11) — keep reductant and ATP vocabulary fresh.

Quick formula strip to memorise: Haber N₂+3H₂→2NH₃; biological N₂+8H⁺+8e⁻+16ATP→2NH₃+16ADP+16Pi; NR NO₃⁻→NO₂⁻ (cytosol); NiR NO₂⁻→NH₃ (plastid); GDH α-KG+NH₃→Glu; transaminase AA₁+keto₂⇌AA₂+keto₁. Say these six lines aloud until automatic. Pair each with one example organism or organelle and you cover most of the short-answer paper for this lesson. Also remember: denitrifying bacteria close the cycle; Chile saltpetre is mineral nitrate; proteins average sixteen percent nitrogen by mass in living organisms.