Biological nitrogen fixation MCQs With Answer is an essential review for B. Pharm students studying microbial ecology, biochemistry, and pharmacognosy. This introduction explains how nitrogenase enzymes, nif genes, symbiotic bacteria (e.g., Rhizobium), and non-symbiotic microbes (e.g., Azotobacter, cyanobacteria) convert atmospheric N2 into biologically usable ammonia. Key concepts include energy (ATP) requirements, oxygen sensitivity, FeMo-cofactor, legume–Rhizobium symbiosis, and assay methods like acetylene reduction and 15N incorporation. Mastering these topics links microbial nitrogen metabolism to amino acid and secondary metabolite synthesis relevant to drug discovery. Now let’s test your knowledge with 50 MCQs on this topic.
Q1. What is the overall stoichiometric reaction for biological nitrogen fixation catalyzed by nitrogenase?
- N2 + 8H+ + 8e- + 16 ATP → 2 NH3 + H2 + 16 ADP + 16 Pi
- N2 + 6H+ + 6e- + 12 ATP → 2 NH3 + 12 ADP + 12 Pi
- N2 + 4H+ + 4e- + 8 ATP → NH3 + H2 + 8 ADP + 8 Pi
- N2 + 10H+ + 10e- + 20 ATP → 2 NH3 + 20 ADP + 20 Pi
Correct Answer: N2 + 8H+ + 8e- + 16 ATP → 2 NH3 + H2 + 16 ADP + 16 Pi
Q2. Which enzyme complex is directly responsible for the reduction of atmospheric N2 to NH3?
- Nitrogenase
- Nitrate reductase
- Nitrite reductase
- Glutamine synthetase
Correct Answer: Nitrogenase
Q3. Which gene encodes the iron (Fe) protein (dinitrogenase reductase) of nitrogenase that provides electrons and ATP coupling?
- nifH
- nifD
- nifK
- vnfH
Correct Answer: nifH
Q4. The FeMo-cofactor, essential for N2 reduction, contains which metal as a central component?
- Molybdenum (Mo)
- Vanadium (V)
- Nickel (Ni)
- Copper (Cu)
Correct Answer: Molybdenum (Mo)
Q5. What is the primary reason nitrogenase is highly sensitive to oxygen?
- Oxygen irreversibly damages Fe-S clusters in nitrogenase
- Oxygen competes with N2 as a substrate for nitrogenase
- Oxygen increases ATP hydrolysis, depleting energy
- Oxygen converts NH3 to N2, reversing fixation
Correct Answer: Oxygen irreversibly damages Fe-S clusters in nitrogenase
Q6. Which protein in legume nodules binds oxygen to protect nitrogenase and maintain low O2 concentration?
- Leghemoglobin
- Hemoglobin
- Myoglobin
- Cytoglobin
Correct Answer: Leghemoglobin
Q7. Which of the following organisms is a classical symbiotic nitrogen fixer associated with legume roots?
- Rhizobium
- Azotobacter
- Clostridium
- Escherichia
Correct Answer: Rhizobium
Q8. Which organism is a free-living (non-symbiotic) nitrogen fixer commonly found in soils?
- Azotobacter
- Frankia
- Bradyrhizobium
- Rhizobium leguminosarum
Correct Answer: Azotobacter
Q9. Which assay is commonly used as an indirect measure of nitrogenase activity by measuring the reduction of acetylene to ethylene?
- Acetylene reduction assay (ARA)
- Bradford protein assay
- Griess reaction
- Kjeldahl nitrogen assay
Correct Answer: Acetylene reduction assay (ARA)
Q10. Which direct method measures biological N2 fixation by tracing incorporation of the heavy isotope into biomass?
- 15N2 incorporation assay
- Acetylene reduction assay
- Nitrate determination
- Ammonia colorimetric assay
Correct Answer: 15N2 incorporation assay
Q11. Which electron carrier often donates electrons to nitrogenase in many diazotrophs?
- Ferredoxin
- NADH dehydrogenase
- Cytochrome c
- Flavin adenine dinucleotide (FAD)
Correct Answer: Ferredoxin
Q12. Which protein subunits compose the Mo-nitrogenase (dinitrogenase) core encoded by nifD and nifK?
- Alpha and beta subunits of MoFe protein
- Gamma and delta subunits of Fe protein
- Alpha and beta subunits of Fe protein
- Single polypeptide monomeric enzyme
Correct Answer: Alpha and beta subunits of MoFe protein
Q13. Which nutrient or compound strongly represses nif gene expression and nitrogenase activity when abundant?
- Ammonium
- Phosphate
- Potassium
- Calcium
Correct Answer: Ammonium
Q14. Which alternative nitrogenase uses vanadium and is expressed under molybdenum deficiency?
- V-nitrogenase (vanadium nitrogenase)
- Fe-only nitrogenase
- Cu-nitrogenase
- Mo-nitrogenase
Correct Answer: V-nitrogenase (vanadium nitrogenase)
Q15. In root nodule symbiosis, which plant-derived signal initiates nodulation in many legumes?
- Flavonoids
- Auxins
- Gibberellins
- Abscisic acid
Correct Answer: Flavonoids
Q16. Which bacterial signal molecule produced by Rhizobium induces nodule formation in legumes?
- Nod factors (lipochitooligosaccharides)
- Peptidoglycan fragments
- Lipopolysaccharides
- Quorum sensing acyl-HSL
Correct Answer: Nod factors (lipochitooligosaccharides)
Q17. Which enzyme assimilates ammonium into glutamine as a first step of nitrogen assimilation?
- Glutamine synthetase (GS)
- Glutamate dehydrogenase (GDH)
- Glutamate synthase (GOGAT)
- Alanine aminotransferase
Correct Answer: Glutamine synthetase (GS)
Q18. Which two-enzyme cycle converts ammonium into glutamate via glutamine for biosynthesis?
- GS-GOGAT cycle
- Glyoxylate cycle
- Urea cycle
- Pentose phosphate pathway
Correct Answer: GS-GOGAT cycle
Q19. Frankia species form nitrogen-fixing symbioses primarily with which group of plants?
- Actinorhizal plants (e.g., Alnus)
- Legumes
- Conifers
- Grasses
Correct Answer: Actinorhizal plants (e.g., Alnus)
Q20. Which environmental factor commonly limits biological nitrogen fixation in soil?
- Oxygen concentration and soil aeration
- Light intensity
- Soil salinity only
- Presence of heavy metals only
Correct Answer: Oxygen concentration and soil aeration
Q21. Which microbial group of cyanobacteria performs nitrogen fixation and often protects nitrogenase in specialized cells called heterocysts?
- Anabaena (heterocystous cyanobacteria)
- Micrococcus
- Streptomyces
- E. coli
Correct Answer: Anabaena (heterocystous cyanobacteria)
Q22. Which cofactor donates molybdenum and iron-sulfur clusters for Mo-nitrogenase catalytic site?
- FeMo-cofactor
- Heme cofactor
- Flavin mononucleotide
- Biotin
Correct Answer: FeMo-cofactor
Q23. Which process in symbiotic nodules supplies the ATP needed for nitrogen fixation?
- Plant-provided carbon metabolism (sucrose to malate) in bacteroids
- Photosynthetic oxygen evolution inside nodules
- Atmospheric methane oxidation
- Uptake of nitrate from soil
Correct Answer: Plant-provided carbon metabolism (sucrose to malate) in bacteroids
Q24. Which molecular oxygen-scavenging mechanism is important in some free-living diazotrophs like Azotobacter?
- High respiratory rate to consume O2 rapidly
- Production of leghemoglobin
- Formation of heterocysts
- Spore formation
Correct Answer: High respiratory rate to consume O2 rapidly
Q25. Which regulatory protein family commonly represses nif gene expression in response to fixed nitrogen?
- NifA/NifL regulatory system
- LacI family repressors
- Two-component Pho regulon
- Heat shock sigma factors
Correct Answer: NifA/NifL regulatory system
Q26. In the context of B.Pharm, why is understanding biological nitrogen fixation relevant to drug discovery?
- Nitrogen availability influences biosynthesis of nitrogenous secondary metabolites used as drugs
- Nitrogen fixation is directly used to synthesize antibiotics in vitro
- All drugs contain nitrogen fixed by nitrogenase only
- It replaces clinical pharmacology knowledge
Correct Answer: Nitrogen availability influences biosynthesis of nitrogenous secondary metabolites used as drugs
Q27. Which measurement indicates production of ethylene from acetylene in an acetylene reduction assay?
- Nitrogenase activity (indirect measure of N2 fixation)
- Respiration rate of plant roots
- Nitrate concentration in soil
- Amount of fixed ammonium in solution
Correct Answer: Nitrogenase activity (indirect measure of N2 fixation)
Q28. Which amino acid is the primary initial product after ammonium is assimilated by GS-GOGAT?
- Glutamine (and subsequently glutamate)
- Alanine
- Asparagine
- Lysine
Correct Answer: Glutamine (and subsequently glutamate)
Q29. What effect does high external nitrate concentration typically have on nitrogen fixation in many diazotrophs?
- Inhibits nitrogenase expression and activity
- Stimulates nitrogenase activity
- No effect on nitrogen fixation
- Converts nitrogenase into nitrate reductase
Correct Answer: Inhibits nitrogenase expression and activity
Q30. Which structural adaptation in legume nodules provides a low-O2 environment while allowing respiration?
- Leghemoglobin-mediated O2 buffering in infected cells
- Development of air pockets inside nodules
- Complete anaerobiosis with no respiration
- Production of oxygen from nitrate reduction
Correct Answer: Leghemoglobin-mediated O2 buffering in infected cells
Q31. Which nitrogenase component directly binds ATP and transfers electrons to the MoFe protein?
- Fe protein (dinitrogenase reductase)
- MoFe protein alpha subunit
- MoFe protein beta subunit
- Cytochrome oxidase
Correct Answer: Fe protein (dinitrogenase reductase)
Q32. Which experimental approach identifies nif gene expression at the mRNA level in diazotrophs?
- RT-PCR of nif transcripts
- Western blot of leghemoglobin
- Kjeldahl nitrogen determination
- Acetylene reduction assay
Correct Answer: RT-PCR of nif transcripts
Q33. Which metal limitation in soils can specifically impair Mo-nitrogenase activity and trigger alternative nitrogenases?
- Molybdenum deficiency
- Iron deficiency only
- Calcium deficiency
- Phosphorus deficiency
Correct Answer: Molybdenum deficiency
Q34. Which gas is produced as a byproduct of nitrogenase activity along with ammonia?
- Hydrogen (H2)
- Methane (CH4)
- Carbon dioxide (CO2)
- Nitrous oxide (N2O)
Correct Answer: Hydrogen (H2)
Q35. Which nitrogen fixation strategy involves bacteria closely associated with plant roots but not forming nodules?
- Associative nitrogen fixation (e.g., Azospirillum)
- Symbiotic nodulation (Rhizobium)
- Endosymbiotic intracellular fixation only
- Mycorrhizal nitrogen fixation
Correct Answer: Associative nitrogen fixation (e.g., Azospirillum)
Q36. Which laboratory condition would most likely increase nitrogenase activity in a culture of Azotobacter?
- Low oxygen, high carbon substrate, low fixed nitrogen
- High oxygen, high ammonium
- No carbon source, high nitrate
- Excess molybdenum with abundant ammonium
Correct Answer: Low oxygen, high carbon substrate, low fixed nitrogen
Q37. nifD and nifK genes encode which components of nitrogenase?
- Alpha and beta subunits of the MoFe protein (dinitrogenase)
- Fe protein subunits
- Leghemoglobin subunits
- Regulatory proteins for nodulation
Correct Answer: Alpha and beta subunits of the MoFe protein (dinitrogenase)
Q38. Which cellular location in legume nodules houses nitrogen-fixing bacteroids?
- Infected plant cells within symbiosomes
- Root epidermis surface
- Xylem vessels exclusively
- Soil pore spaces outside root
Correct Answer: Infected plant cells within symbiosomes
Q39. What role does the plant supply of malate play in nodule bacteroids?
- Provides carbon and reducing power for respiration and ATP production
- Acts as a direct nitrogen donor to the bacteria
- Oxidizes nitrogenase to activate it
- Removes leghemoglobin from nodules
Correct Answer: Provides carbon and reducing power for respiration and ATP production
Q40. Which is a direct genetic marker commonly used to detect diazotrophs in environmental samples?
- nifH gene sequences
- 16S rRNA sequences only
- rbcL gene sequences
- cytochrome b sequences
Correct Answer: nifH gene sequences
Q41. Which statement best describes the ecological significance of biological nitrogen fixation?
- It converts inert atmospheric N2 into bioavailable forms supporting primary productivity
- It removes nitrogen from ecosystems permanently
- It primarily produces nitrate for plant uptake
- It is insignificant compared to industrial fixation in natural ecosystems
Correct Answer: It converts inert atmospheric N2 into bioavailable forms supporting primary productivity
Q42. Which compound is commonly formed when nodules export fixed nitrogen to the plant in some legumes?
- Amides like asparagine or ureides depending on species
- Free atmospheric N2
- Elemental nitrogen (N0)
- Nitrite exclusively
Correct Answer: Amides like asparagine or ureides depending on species
Q43. Which microbial enzyme is involved in protecting nitrogenase from oxidative damage by detoxifying reactive oxygen species inside cells?
- Catalase/peroxidase systems
- Urease
- Nitrate reductase
- Reverse transcriptase
Correct Answer: Catalase/peroxidase systems
Q44. Which feature distinguishes heterocystous cyanobacteria’s approach to nitrogen fixation?
- Spatial separation: heterocysts provide microoxic sites for nitrogenase
- They use leghemoglobin to bind O2
- They form root nodules with plants
- They perform nitrogen fixation only in darkness
Correct Answer: Spatial separation: heterocysts provide microoxic sites for nitrogenase
Q45. Which of the following best describes the role of nitrogen fixation in pharmaceutical natural product biosynthesis?
- Fixed nitrogen provides building blocks (amino groups) for alkaloids and other N-containing drugs
- Nitrogen fixation synthesizes synthetic antibiotics directly
- All plant alkaloids derive from atmospheric N2 without metabolism
- It only affects vitamin D synthesis in plants
Correct Answer: Fixed nitrogen provides building blocks (amino groups) for alkaloids and other N-containing drugs
Q46. Which factor would most likely upregulate nif gene expression in free-living azotrophs?
- Low combined nitrogen (e.g., low ammonium or nitrate)
- High ammonium concentration
- High soil nitrate concentration
- Excess oxygen and no carbon
Correct Answer: Low combined nitrogen (e.g., low ammonium or nitrate)
Q47. Which product of nitrogenase is often recycled or oxidized by microbes, representing an energy loss during fixation?
- Hydrogen gas (H2)
- Methane (CH4)
- Carbon monoxide (CO)
- Nitrous oxide (N2O)
Correct Answer: Hydrogen gas (H2)
Q48. Which laboratory technique can quantify ammonium produced from N2 fixation after incubation?
- Colorimetric ammonia assay (e.g., Nessler’s or phenol-hypochlorite)
- Gram staining
- Thin-layer chromatography of sugars
- DNA gel electrophoresis
Correct Answer: Colorimetric ammonia assay (e.g., Nessler’s or phenol-hypochlorite)
Q49. Which statement about associative diazotrophs like Azospirillum is true?
- They enhance plant growth by fixing N near roots and producing phytohormones
- They only fix nitrogen inside root nodules
- They are obligate anaerobes incapable of surviving oxygen
- They always convert all fixed N into atmospheric N2
Correct Answer: They enhance plant growth by fixing N near roots and producing phytohormones
Q50. Which strategy is being researched to reduce reliance on chemical fertilizers by leveraging biological nitrogen fixation for non-legume crops?
- Engineering associative or symbiotic N-fixation into cereals and improving diazotroph associations
- Increasing industrial Haber-Bosch fertilizer application
- Eliminating microbial communities from rhizospheres
- Applying high doses of nitrate to soils
Correct Answer: Engineering associative or symbiotic N-fixation into cereals and improving diazotroph associations
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