acetic acid and vinegar production
DESCRIPTION
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 : - PowerPoint PPT PresentationTRANSCRIPT
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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
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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)
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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
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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
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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
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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?
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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)
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TCA Cycle – Electron and Carbon FlowCitric acid synthesis during trophophase
246
82
103
104 186
186
165144
124
124
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?
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TCA Cycle – Metabolites82
103
104186
165
144
124
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?
124
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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
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TCA Cycle – Metabolites82
103
104
186
165
144
124
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
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TCA Cycle – Electron and Carbon FlowCitric acid synthesis during idiophase
246
82
103
104 186
186
165144
124
124
Glucose
Pyruvate
Acetyl-CoA
Citrate
Isocitrate
α-ketoglutarate
OAA
Malate
Fumarate
Succinate
glycolysis
Citrate synthase
01Pyruvatecarboxylase
103 10401 →+ + 82Pyruvate + CO2 + Acetyl-CoA → Citrate
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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))?
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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
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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+→
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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?
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• 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)
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TCA Cycle – Electron and Carbon FlowCitric acid synthesis during trophophase
82
104 186
186
165144
124
124
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?
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Glyoxylate Formation from Isocitrate Lyase
82
104 186
186
Acetyl-CoA
Citrate
Isocitrate
OAA Citrate synthase
42
Citric Acid Synthesis With Lipids as the Substrate
Isocitratelyase
Aconitase
Glyoxylate(CHO-COOH)
144
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Glyoxylate use to lead to OAA via malate
82
104 186
186
Acetyl-CoA
Citrate
Isocitrate
OAA Citrate synthase
42
Citric Acid Synthesis With Lipids as the Substrate
Isocitratelyase
Aconitase
Glyoxylate(CHO-COOH)
144
124
82
Malate
How can the excretion of citrate be guaranteed when isocitrateis necessary for citrate synthesis?
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(Glyoxylate Cycle)
82
104 186
186
144
124
124
Acetyl-CoA
Citrate
Isocitrate
OAAMalate
Fumarate
Succinate
Citrate synthase
42
Citric Acid Synthesis With Lipids as the Substrate
Isocitratelyase
Aconitase
82
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.
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(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
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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
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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
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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
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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
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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
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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
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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
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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%
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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:
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Glutamate Production 1246
82
103
104 186
186
165144
124
124
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
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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)
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Glutamate Production 1246
82
103
104 186
186
165144
124
124
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
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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
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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
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• 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)