A. Fundamentals of Organic Acids
1. What is the difference between Brønsted–Lowry and Lewis acids?
Brønsted–Lowry acids donate protons (H⁺), while Lewis acids accept electron pairs to form covalent bonds.
2. What are Ka and pKa, and how do they relate to acid strength?
Ka measures the degree of dissociation of an acid; pKa = −log₁₀(Ka). Lower pKa means a stronger acid.
3. What are monoprotic and polyprotic acids?
Monoprotic acids donate one proton (H⁺) per molecule; polyprotic acids can donate more than one (diprotic, triprotic, etc.).
4. How are organic acids produced in microorganisms?
In anaerobes, acids regenerate NAD⁺ for glycolysis (e.g., lactic acid). In aerobes/fungi, acids form when nutrients are limited, causing incomplete oxidation.
5. Give examples of organic acids produced by microbes.
Citric, itaconic, gluconic, malic, acetic, lactic, oxalic acids.
B. Citric Acid Production
6. What is the IUPAC name of citric acid?
2-hydroxy-propane-1,2,3-tricarboxylic acid.
7. What are its pKa values and buffering range?
pKa₁ = 3.1, pKa₂ = 4.7, pKa₃ = 6.4; buffer capacity between ~2–7.
8. Why does citric acid reduce sweetness?
Citric acid lowers the pH around sweet taste receptors, making them less sensitive to sweet substances, so the sweetness is perceived as weaker.
9. How does citric acid act as an antioxidant and preservative?
Chelates metal ions (Fe²⁺, Cu²⁺), prevents oxidation, stabilizes color and flavor, and inhibits bacterial growth.
10. What are the main applications of citric acid?
Used in food (acidulant, preservative), pharmaceuticals, cleaning agents, and metal treatment.
11. Describe the historical milestones in citric acid discovery and production.
First isolated by Scheele (1784) from lemon; industrial extraction in 1860 (England); Aspergillus niger identified as producer (Currie, 1917).
12. What are the three main fermentation methods for citric acid?
Surface fermentation, solid-state fermentation, and submerged fermentation (most common today).
13. Explain the surface fermentation process.
Large shallow trays with liquid medium inoculated with spores; fungus forms a mat and secretes citric acid; simple but labor-intensive.
14. Why is Aspergillus niger an ideal producer?
It tolerates low pH (1.5–2), converts glucose almost quantitatively, and grows well under nutrient limitations.
15. What is the stoichiometric equation for citric acid formation?
C₆H₁₂O₆ + 0.5O₂ → C₆H₈O₇ + 4H⁺
16. Why is oxygen supply critical in citric acid fermentation?
Because oxygen is the electron acceptor in aerobic oxidation; it regulates redox balance and product formation.
17. Describe the role of manganese (Mn²⁺) in citric acid accumulation.
Extremely low Mn²⁺ levels (<5 ppb) are required. Higher Mn²⁺ inhibits citric acid synthesis and changes cell morphology.
18. What is the role of phosphofructokinase in citric acid biosynthesis?
It regulates glycolysis; inhibited by ATP and citrate but activated by NH₄⁺, AMP, and F-2,6-bisphosphate.
19. Explain how morphology affects citric acid production.
Mn²⁺ deficiency causes small, dense pellets that improve oxygen transfer and mass flow, leading to higher yields.
20. List optimal fermentation conditions for *A. niger* citric acid production.
High sugar (15%), low N and phosphate, pH < 3, temperature 30–35°C, low metals (especially Mn²⁺), and high aeration.
21. What is the role of the alternative oxidase pathway?
Allows electron transfer without ATP production; maintains NADH oxidation and redox balance under limited energy yield.
22. How is citric acid recovered from broth?
By precipitation with Ca(OH)₂ to form calcium citrate, then treated with H₂SO₄ to release citric acid and remove CaSO₄ (gypsum).
23. What is the drawback of the calcium citrate method?
It produces about 16 tons of gypsum waste per ton of citric acid.
24. What are advantages of solvent extraction in recovery?
Reduces waste and allows selective recovery using immiscible solvents (esters, alcohols, amines).
25. How does *Yarrowia lipolytica* differ from *A. niger*?
Yeast grows at pH ≈ 5, insensitive to Mn²⁺, produces citric acid under nitrogen limitation, and can use n-alkanes or glucose.