Acetic Acid and Vinegar Production

1. Acetic Acid and Vinegar Production History • As old as wine making (10,002 y) • Hannibal Uses: • Food acid and preservative, • medical agent • Volatile (not for cooking) Biochemistry Aerobic incomplete oxidation of organics to acetic acid TCA cycle not fully operating Substrates: Ethanol, glucose, hydrocarbons

2. -4 -4 8 8 2 0 2 2 10 10 12 12 0 0 0 2 0 0 0 2 2 2 2 2 ETP Acetic Acid and Vinegar Production = CH3-CH2OH = 2 red. equiv. = CH3-CH2O = O2 = CH3-COOH 6 ATP Bacteria Underoxidiser: Gluconobacter Overoxidiser: Acetobacter (can totally oxidise to CO2)

3. Acetic Acid and Vinegar Production Processes Leave wine open to air → surface process Trickling generator with wood shavings Submersed process (CSTR) + more economic - Lower taste quality Wood Shavings

4. Acetic Acid and Vinegar Production Downstream Only filtering to remove biomass Critical process conditions: • 30°C (Cooling required for CSTR) • Maximum ETOH concentration: 13% 50% inactive cells after 1 min air off due to acetaldehyde accumulation ↑ [etOH] + ↑ [acetic acid] + ↓ [O2] → ↑ acetaldehyde Product yield (g ac./ g etOH): up to 98%

5. Citric Acid Production Special properties: Complexing agent for metals (Fe, Ca) Uses: • Principle food acid in soft drinks, jams • Food preservative • Medical: iron citrate as iron supplement anticoagulant for storage of blood • Detergent to replace phosphorus thus avoiding eutrophication • Used in metal cleaning fluid • Used as siderphore by microbes Fe(OH)3 + citrate → Fe3+ - citrate complex (not available for uptake by cells) → bio-available

6. Citric Acid Production Biochemistry TCA cycle, Glyoxylate cycle Gaden’s fermentation type II • Trophophase: growth and complete substrate oxidation to CO2 • Idiophase: deregulated TCA cycle due to iron limitation: ↓↓α-ketoglutarate DH, ↓ Aconitase ↓ Isocytrate lyase, ↑ Citrate synthase. Why?

7. 0 10 10 1 3 4 Citric Acid Production Reasons for citrate excretion: 1. Aconitase contains an iron sulfur centre Thus Fe limitation → citrate conversion inhibited 2. Citrate is a siderophore Thus iron limitation can be expected to stimulate citrate synthase Problem: Citrate excretion → interruption of TCA cycle → no more OAA, citrate excretion ceases Solution: Pyruvate carboxylase (key enzyme for citric acid production): Pyruvate + CO2 → OAA → + Anaplerotic sequences to replenish reactions of TCA cycle (usually for biosynthesis)

8. 8 16 18 12 10 18 14 24 12 10 2 6 4 5 4 6 3 6 4 4 TCA Cycle – Electron and Carbon Flow Citric acid synthesis during trophophase Glucose glycolysis Pyruvate Acetyl-CoA Citrate OAA Citrate synthase Malate DH Aconitase Malate Isocitrate Fumarase Fumarate Isocitrate DH Succinate DH α-ketoglutarate Succinate α-ketoglutarate DH How can the cycle continue when citrate is excreted?

9. 8 10 10 12 18 12 16 14 2 5 3 4 6 4 4 4 TCA Cycle – Metabolites Acetyl-CoA CH2-COOH Citrate COH-COOH CH2-COOH OAA HOOC-CO-CH2-COOH α-ketoglutarate HOOC-CH2-CH2-CO-COOH 1-6-6-2-1 Fumarate HOOC-CH=CH-COOH 1-5-5-1 Succinate HOOC-CH2-CH2-COOH 1-6-6-1 Malate HOOC-CH2-CHOH-COOH 1-6-4-1 Pyruvate CH3-CO-COOH How can the cycle continue when citrate is excreted?

10. TCA Cycle – Citrate isomerisation CH2 - COOH | Citrate HOCOH -COOH | CH2 - COOH CH2 - COOH | Iso-Citrate CH - COOH | HOCH - COOH CH2 - COOH | cis-Aconitate CH - COOH || HOCH - COOH

11. 8 10 10 12 18 12 16 14 2 5 3 4 6 4 4 4 TCA Cycle – Metabolites Acetyl-CoA OAA HOOC-CO-CH2-COOH 1-2-6-1 CH2-COOH Citrate 1-6-3-1-6-1 COH-COOH CH2-COOH α-ketoglutarate HOOC-CH2-CH2-CO-COOH 1-6-6-2-1 Fumarate HOOC-CH=CH-COOH 1-5-5-1 Succinate HOOC-CH2-CH2-COOH 1-6-6-1 Malate HOOC-CH2-CHOH-COOH 1-6-4-1 Pyruvate CH3-CO-COOH 7-2-1

12. 0 8 8 0 10 24 10 10 12 10 16 14 12 18 18 2 1 2 1 4 4 6 3 6 6 5 4 4 4 3 TCA Cycle – Electron and Carbon Flow Citric acid synthesis during idiophase Glucose glycolysis Pyruvate Pyruvate carboxylase Acetyl-CoA Citrate OAA Citrate synthase Malate Isocitrate Fumarate α-ketoglutarate Succinate → + + Pyruvate + CO2 + Acetyl-CoA → Citrate

13. TCA Cycle – Electron and Carbon Flow Citric acid synthesis during idiophase 1 mol glucose can result in 1 mol citric acid! 6 electrons need to be disposed of (oxygen) How can citrate be synthesised when pyruvate is not available (e.g. when lipids are the substrate (ß-oxidation))?

14. Citric Acid Synthesis With Lipids as the Substrate • Aim: Produce citrate from non-carbohydrate material • e.g.: hydrocarbons, fatty acids, ethanol, acetate • Problem: ß-oxidation rather than glycolysis is used • pyruvate (Pyr carbox.) not available for OAA synthesis • Solution: Glyoxylate cycle • designed to convert fat into carbohydrates (C2->C3) • plant seedlings, microbes, but not animals

15. 4 2 8 12 10 0 2 2 4 4 → → + + Acetate + Glyoxylate → Malate → OAA + 2 NADH Citric Acid Synthesis With Lipids as the Substrate • Glyoxylate (COH-COOH): • is the second most oxidised biological organic substance • can be fused with acetate to lead to OAA • OAA can then be used for the generation of new citrate • What is the reaction that forms glyoxylate ? • Can you think what is the most oxidised organic ?

16. 4 12 18 2 4 6 Citric Acid Synthesis With Lipids as the Substrate Glyoxylate is derived from isocitrate lyase reaction: (see glyoxylate cycle) → + Isocitrate → Succinate + Glyoxylate How can the excretion of citrate be guaranteed when isocitrate is necessary for citrate synthesis?

17. Example calculation: • Bioreactor: steady state at DO 2 mg/L assume the sat conc to be 8 mg/L • stopped the airflow  • OUR = 200 mg/L/h • What would be the max oxidation rate of acetate to CO2 by the reactor when the DO must be at least 1 mg/L? • steady state  OUR = OTR • kLa = OTR /(cs – cL) = 200 mg/L/h /(8-2 mg/L)= 33.3 h-1 • OTR at cL = 1 mg/L is OTR = kLa * (8 – 1 mg/L) =233 mg/L/h = 7.3 mmol/L/h  • 3.65 mmol of acetate can be oxidised when the reactor runs at DO of 1 mg/L • (MW 32 g/mol)

18. 8 12 10 18 18 16 14 12 2 4 4 6 6 5 4 4 TCA Cycle – Electron and Carbon Flow Citric acid synthesis during trophophase Acetyl-CoA Citrate OAA Citrate synthase Malate DH Aconitase Malate Isocitrate Fumarase Fumarate Isocitrate DH Succinate DH α-ketoglutarate Succinate α-ketoglutarate DH How can the cycle continue when citrate is excreted?

19. 8 4 10 18 18 14 2 2 6 6 4 4 Citric Acid Synthesis With Lipids as the Substrate Glyoxylate Formation from Isocitrate Lyase Acetyl-CoA Citrate OAA Citrate synthase Aconitase Isocitrate Isocitrate lyase Glyoxylate (CHO-COOH)

20. 4 8 8 10 14 18 18 12 2 2 2 4 6 4 4 6 Citric Acid Synthesis With Lipids as the Substrate Glyoxylate use to lead to OAA via malate Acetyl-CoA Citrate OAA Citrate synthase Aconitase Malate Isocitrate Isocitrate lyase Glyoxylate (CHO-COOH) How can the excretion of citrate be guaranteed when isocitrate is necessary for citrate synthesis?

21. 4 8 8 10 18 18 14 12 12 2 2 2 4 6 4 4 6 4 Citric Acid Synthesis With Lipids as the Substrate (Glyoxylate Cycle) Acetyl-CoA Citrate OAA Citrate synthase Aconitase Malate Malate synthase Isocitrate Fumarate Isocitrate lyase Succinate Glyoxylate (CHO-COOH) Isocitrate supplies precursors (succinate and glyoxylate) for two OAA,  thus allowing the synthesis of 2 citrate,  one to be excreted, the second to continue the glyox. cycle.

22. 6 8 18 0 2 6 Citric Acid Synthesis With Lipids as the Substrate (Glyoxylate Cycle) Glyoxylate cycle can produce citrate from acetate only: 3 → + 3 Acetate → Citrate + 6 H (3 NADH) And again, from the balance we can see that an electron acceptor is needed to accept the excess electrons

23. Citric Acid Production - Process Conditions • • Citrate is not a primary metabolite • Not formed during exponential growth • but under Fe limitation • Continuous chemostat culture not suitable • Virtual absence of Fe is important: • ↑ Fe3+ → ↓ [citric acid], ↑ [oxalic acid], CO2 • No iron vessels (not even stainless steel) • Addition of Cu and Zn salts as iron antagonist • Organisms: • Aspergillus niger on sugar media • Candida yeast on alcanes: • pH must be less than 3.5, otherwise oxalate excretion

24. 2 4 2 0 2 2 Citric Acid Production Industrial Problems •Possible reaction of oxalic acid production: → + Glyoxylate → Oxylate + NADH Is anaerobic citric acid production from fats or glucose likely? What is the expected difference in biomass formation during tropho- and idio- phase ? (3ATP/NADH oxidised = 6ATP/O2 used) Interesting biochem: Why is it possible to increase the citric acid output of a glucose degrading culture of A. niger by adding hydrocarbons as a supplement? PEP inhib. ICL phosphoenolpyruvate inhibits isocitratelyase for good reason: If PEP is there then there is no need to run glyoxylate cycle

25. Citric Acid Production Process History: • First extracted from immature lemons • 1883 shown microbial metabolite • 1922 nutrient deficiency (Fe) was found to result in high [citrate] Strain: Aspergillus niger mutants Submerged process (airlift or CSTR) • pellets formation • requires well cultivated seed material • high productivity, low labour costs • high capital costs, foaming problems

26. Citric Acid Production Process Open vats (still used, cheaper O2 supply) • blow spores onto medium in high purity aluminium vats • allow white mycelium to grow • after pH 5 → 2, drain off liquid and renew (2nd idiophase!) • low capital, high labour costs (Australia) Koji fermentation – Solid surface process (Japan) • similar to shallow trickling filter • support material (wheat bran, etc.) • lower sensitivity of Fe

27. Citirc Acid Production Process Critical process conditions: • Medium: 15 – 25% sucrose solutions (molasses, starch hydrolysates) • 2mg/L Fe3+ required in trophophase • Less than 0.1 mg/L Fe3+ desired in idiophase • Startup pH 5 → drops to pH 2 → low risk of contamination

28. 24 22 6 6 Gluconic Acid Production Process Special property: Complex Ca2+ and Mg2+ ions Use: • Ca gluconate as soluble Ca medication • Sequestering agent in neutral or alkaline solutions E.g. Bottle washing (removes Ca precipitates) • Gluconolactone has latent acidogenic properties Heating gluconolactone →↓ pH because of gluconic acid production (e.g. baking powder, self raising flour) Biochemistry: Glucose oxidation by oxygen with glucose oxidase (biosensors) Glucose + O2 → Gluconate + H2O2 →

29. Gluconic Acid Production Process •Strain: Aspergillus niger • Acetobacter suboxidans (also oxidises other alcohol groups to organic acids (e.g. propanol to propionate) → bioconversions Process: submersed Critical process conditions • glucose medium • low temperature (20 °C) • N limitation • neutral pH • absolute sterility

30. Amino Acid Production Glutamate Glutamate and lysine are the most significant commercial amino acids produced by bioprocesses. Strong competition existing from: • chemical synthesis • extraction from animal protein Glutamate is the only mass product Rest: 2% Lysine: 11% Glutamate: 87% Use: Food additive (“flavour enhancer”) Japan, China,… Sold as mono-sodium-glutamate (MSG) Has had bad reputation because of over use.

31. Amino Acid Production Glutamate Biochemistry: • Glycolysis, TCA cycle • reductive amination of α-ketoglutarate (glutamate DH) • block α-ketoglutarate DH • accumulation of α-ketoglutarate • under excess of NH3 → glutamate accumulation • accumulation of glutamate and thus α-ketoglutarate removal requires an anaplerotic sequence to replenish TCA cycle:

32. 8 2 24 10 10 18 18 14 12 12 16 18 2 0 4 5 4 6 4 5 6 3 6 4 Glutamate Production 1 Glucose glycolysis Pyruvate Acetyl-CoA Citrate OAA Citrate synthase Malate DH Aconitase Malate Isocitrate Fumarase Fumarate Isocitrate DH Succinate DH α-ketoglutarate Succinate α-ketoglutarate DH Glutamate DH NH3 Glutamate N

33. 2 0 12 10 0 1 4 3 Amino Acid Production Glutamate • accumulation of glutamate and thus α-ketoglutarate removal requires an anaplerotic sequence to replenish TCA cycle: Malic enzyme: Pyruvate + 2 H + CO2 → Malate + + → With hydrocarbons as the substrate: glyoxylate cycle is operable (refer to citric acid production)

34. 0 2 8 2 18 10 24 16 10 18 12 12 14 18 0 1 0 2 6 6 4 5 4 4 4 3 5 6 Glutamate Production 1 Glucose glycolysis Pyruvate Malic Enzyme Acetyl-CoA Citrate OAA Citrate synthase Malate DH Aconitase Malate Isocitrate Fumarase Fumarate Isocitrate DH Succinate DH α-ketoglutarate Succinate α-ketoglutarate DH Glutamate DH NH3 Glutamate N

35. 6 0 24 18 0 1 5 6 Glutamate Production 2 (Feedback inhibition) Glucose + NH3 → Glutamate + CO2 + 6H + N N + + Problem: • glutamate accumulates in the cell causing feedback inhibition (glutamate is not meant to be endproduct (no excretion mechanism)) • Weakened cell membranes are required • Weak membranes are low in unsaturated phospholipids. This can be achieved by: •Biotin deficiency (complex media can not be used) •Addition of saturated fatty acid •Addition of sub lethal doses of penicillin

36. Organisms: • Usually Corynebacterium glutamicium, however • no specific group as long as blocked at a-ketoglutarate DH • Oleate or glycerol auxotrophic mutants used. Growth in the presence of low concentrations of glycerol or oleate Process: • 160 g/L of glucose or acetate medium • pH neutral –>( very prone to contamination) • batch process (revertants (“contamination from inside”, phages, contamination) • 2 -4 days of duration in • submersed process (CSTR) • high oxygen requirement (high KLA) necessary • cooling necessary

37. combined pH control by NH3 addition allows: • • to optimise N-supply, • • to monitor amino acid production by NH3 used Low oxygen concentration can result in succinate or lactate production (pyruvate hydrogenation)