Acetic Acid and Vinegar Production
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Transcript of Acetic Acid and Vinegar Production
Acetic Acid and Vinegar ProductionHistory• As old as wine making (10,002 y)• Hannibal
Uses:• Food acid and preservative, • medical agent• Volatile (not for cooking)
BiochemistryAerobic incomplete oxidation of organics to acetic acidTCA cycle not fully operating
Substrates: Ethanol, glucose, hydrocarbons
10220
122 12220102
= CH3-CH2OH= 2 red. equiv.= CH3-CH2O
-40
82
20
6 ATP
00ETP -40 = O282 = CH3-COOH
Acetic Acid and Vinegar Production
BacteriaUnderoxidiser: GluconobacterOveroxidiser: Acetobacter (can totally oxidise to CO2)
Acetic Acid and Vinegar Production
Woo
dS
havi
ngs
Processes Leave wine open to air→ surface process
Trickling generator with wood shavings
Submersed process (CSTR)+ more economic- Lower taste quality
DownstreamOnly 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 acetaldehydeaccumulation↑ [etOH] + ↑ [acetic acid] + ↓ [O2] → ↑ acetaldehydeProduct yield (g ac./ g etOH): up to 98%
Acetic Acid and Vinegar Production
Citric Acid ProductionSpecial 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
Citric Acid ProductionBiochemistry
TCA cycle, Glyoxylate cycleGaden’s fermentation type II
• Trophophase: growth and complete substrate oxidationto CO2
• Idiophase: deregulated TCA cycle due to iron limitation:
↓↓α-ketoglutarate DH, ↓ Aconitase ↓ Isocytrate lyase,↑ Citrate synthase. Why?
Citric Acid ProductionReasons for citrate excretion:1. Aconitase contains an iron sulfur centre
Thus Fe limitation → citrate conversion inhibited2. Citrate is a siderophore
Thus iron limitation can be expected to stimulatecitrate 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 → OAA103 10401 →+
Anaplerotic sequences to replenish reactions of TCA cycle(usually for biosynthesis)
TCA Cycle – Electron and Carbon FlowCitric acid synthesis during trophophase
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Glucose
Pyruvate
Acetyl-CoA
Citrate
Isocitrate
α-ketoglutarate
OAA
Malate
Fumarate
Succinate
α-ketoglutarate DH
glycolysis
Citrate synthaseAconitase
Isocitrate DHSuccinate DH
Fumarase
Malate DH
How can the cycle continue when citrate is excreted?
TCA Cycle – Metabolites82
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104186
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Pyruvate CH3-CO-COOH
Acetyl-CoA
CH2-COOHCitrate COH-COOH CH2-COOH
α-ketoglutarate HOOC-CH2-CH2-CO-COOH 1-6-6-2-1
OAA HOOC-CO-CH2-COOH
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
How can the cycle continue when citrate is excreted?
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TCA Cycle – Citrate isomerisation
CH2 - COOH |
Citrate HOCOH -COOH |CH2 - COOH
CH2 - COOH |
cis-Aconitate CH - COOH ||
HOCH - COOH
CH2 - COOH |
Iso-Citrate CH - COOH |
HOCH - COOH
TCA Cycle – Metabolites82
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186
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Pyruvate CH3-CO-COOH 7-2-1
Acetyl-CoA
CH2-COOHCitrate 1-6-3-1-6-1 COH-COOH CH2-COOH
α-ketoglutarate HOOC-CH2-CH2-CO-COOH 1-6-6-2-1
OAA HOOC-CO-CH2-COOH 1-2-6-1
Fumarate HOOC-CH=CH-COOH 1-5-5-1
Succinate HOOC-CH2-CH2-COOH 1-6-6-1
Malate HOOC-CH2-CHOH-COOH 1-6-4-1124
TCA Cycle – Electron and Carbon FlowCitric acid synthesis during idiophase
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Glucose
Pyruvate
Acetyl-CoA
Citrate
Isocitrate
α-ketoglutarate
OAA
Malate
Fumarate
Succinate
glycolysis
Citrate synthase
01Pyruvatecarboxylase
103 10401 →+ + 82Pyruvate + CO2 + Acetyl-CoA → Citrate
TCA Cycle – Electron and Carbon FlowCitric 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))?
Citric Acid Synthesis With Lipids as the Substrate
Aim: Produce citrate from non-carbohydrate materiale.g.: hydrocarbons, fatty acids, ethanol, acetate
Problem: ß-oxidation rather than glycolysis is usedpyruvate (Pyr carbox.) not available for OAA synthesis
Solution: Glyoxylate cycledesigned to convert fat into carbohydrates (C2->C3)plant seedlings, microbes, but not animals
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 ?
Citric Acid Synthesis With Lipids as the Substrate
12410442 →+82
Acetate + Glyoxylate → Malate → OAA + 2 NADH
20+→
Glyoxylate is derived from isocitrate lyase reaction:
Citric Acid Synthesis With Lipids as the Substrate
124 42+Isocitrate → Succinate + Glyoxylate
→186 (see glyoxylate cycle)
How can the excretion of citrate be guaranteed when isocitrateis necessary for citrate synthesis?
• 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)
TCA Cycle – Electron and Carbon FlowCitric acid synthesis during trophophase
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Acetyl-CoA
Citrate
Isocitrate
α-ketoglutarate
OAA
Malate
Fumarate
Succinate
α-ketoglutarate DH
Citrate synthaseAconitase
Isocitrate DHSuccinate DH
Fumarase
Malate DH
How can the cycle continue when citrate is excreted?
Glyoxylate Formation from Isocitrate Lyase
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104 186
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Acetyl-CoA
Citrate
Isocitrate
OAA Citrate synthase
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Citric Acid Synthesis With Lipids as the Substrate
Isocitratelyase
Aconitase
Glyoxylate(CHO-COOH)
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Glyoxylate use to lead to OAA via malate
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104 186
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Acetyl-CoA
Citrate
Isocitrate
OAA Citrate synthase
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Citric Acid Synthesis With Lipids as the Substrate
Isocitratelyase
Aconitase
Glyoxylate(CHO-COOH)
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Malate
How can the excretion of citrate be guaranteed when isocitrateis necessary for citrate synthesis?
(Glyoxylate Cycle)
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Acetyl-CoA
Citrate
Isocitrate
OAAMalate
Fumarate
Succinate
Citrate synthase
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Citric Acid Synthesis With Lipids as the Substrate
Isocitratelyase
Aconitase
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Malate synthase
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.
(Glyoxylate Cycle)Citric Acid Synthesis With Lipids as the Substrate
Glyoxylate cycle can produce citrate from acetate only:
60+
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
→ 186823
Citric Acid Production - Process Conditions
• Citrate is not a primary metaboliteNot formed during exponential growthbut under Fe limitationContinuous chemostat culture not suitable
Virtual absence of Fe is important:↑ Fe3+ → ↓ [citric acid], ↑ [oxalic acid], CO2No iron vessels (not even stainless steel)Addition of Cu and Zn salts as iron antagonist
Organisms:Aspergillus niger on sugar mediaCandida yeast on alcanes:pH must be less than 3.5, otherwise oxalate excretion
Citric Acid Production Industrial Problems
•Possible reaction of oxalic acid production:
20+
Glyoxylate → Oxylate + NADH
→42 22
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. ICLphosphoenolpyruvate inhibits isocitrate lyase for good reason: If PEP is there then there is no need to run glyoxylate cycle
Citric Acid Production ProcessHistory:• 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
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
Citric Acid Production Process
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
Gluconic Acid Production ProcessSpecial property:Complex Ca2+ and Mg2+ ionsUse:• Ca gluconate as soluble Ca medication• Sequestering agent in neutral or alkaline solutionsE.g. Bottle washing (removes Ca precipitates)• Gluconolactone has latent acidogenic propertiesHeating gluconolactone →↓ pH because of gluconic acidproduction (e.g. baking powder, self raising flour)
Biochemistry:Glucose oxidation by oxygen with glucose oxidase (biosensors)
Glucose + O2 → Gluconate + H2O2→246 226
Gluconic Acid Production Process•Strain:Aspergillus niger• Acetobacter suboxidans (also oxidises other alcohol groupsto 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
Amino Acid ProductionGlutamate
Glutamate and lysine are the most significant commercialamino acids produced by bioprocesses.
Strong competition existing from:• chemical synthesis • extraction from animal proteinGlutamate is the only mass product
Use: Food additive (“flavour enhancer”) Japan, China,…Sold as mono-sodium-glutamate (MSG)Has had bad reputation because of over use.
Glutamate: 87%
Rest: 2%Lysine: 11%
Amino Acid ProductionGlutamate
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 removalrequires an anaplerotic sequence to replenish TCA cycle:
Glutamate Production 1246
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Glucose
Pyruvate
Acetyl-CoA
Citrate
Isocitrate
α-ketoglutarate
OAA
Malate
Fumarate
Succinate
α-ketoglutarate DH
glycolysis
Citrate synthaseAconitase
Isocitrate DHSuccinate DH
Fumarase
Malate DH
185 N
20NH3Glutamate
Glutamate DH
Amino Acid ProductionGlutamate
• accumulation of glutamate and thus α-ketoglutarate removalrequires an anaplerotic sequence to replenish TCA cycle:
Malic enzyme:
20+Pyruvate + 2 H + CO2 → Malate
→ 12401103 +
With hydrocarbons as the substrate: glyoxylate cycle isoperable (refer to citric acid production)
Glutamate Production 1246
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104 186
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Glucose
Pyruvate
Acetyl-CoA
Citrate
Isocitrate
α-ketoglutarate
OAA
Malate
Fumarate
Succinate
α-ketoglutarate DH
glycolysis
Citrate synthaseAconitase
Isocitrate DHSuccinate DH
Fumarase
Malate DH
185 N
20NH3Glutamate
Glutamate DH
2001
Malic Enzyme
Glutamate Production 2(Feedback inhibition)
Glucose + NH3 → Glutamate + CO2 + 6H
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
185 01N + 60246 ++ N
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
• 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)