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

102

20

12212220

102

= 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 Production

BiochemistryTCA 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 Production

Reasons for citrate excretion:

1. Aconitase contains an iron sulfur centreThus Fe limitation → citrate conversion inhibited

2. 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 → OAA

103 10401 →+Anaplerotic sequences to replenish reactions of TCA cycle(usually for biosynthesis)

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?

TCA Cycle – Metabolites

82

103

104

186

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

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 – Metabolites

82

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

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

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

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?

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

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?

(Glyoxylate Cycle)

82

104 186

186

144

124

124

Acetyl-CoA

Citrate

Isocitrate

OAA

Malate

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.

(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 Industrial Problems

• Citrate is not a primary metaboliteNot formed during exponential growthbut under Fe limitationContinuous chemostat culture not suitableunless as multitank system

↑ Na+ → yellow pigment and oxalic acid production• ↑ Fe3+ → ↓ [citric acid], ↑ [oxalic acid], CO2No iron vessels (not even stainless steel)• Addition of Cu and Zn salts as iron antagonist

Typically using Aspergillus niger on sugar media• Use of alcanes and Candida yeast as biocatalyst:+ ↑ product yields-low sloubility of substrate (↓ production rate R)-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

Citirc Acid Production Process

Strain: Aspergillus niger mutants

History:• First extracted from immature lemons• 1883 shown microbial metabolite

• 1922 nutrient deficiency (Fe) was found to result in high [citrate]

Process:

• 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+ ions

Use:• 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 ProcessStrain:• 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 removal

requires an anaplerotic sequence to replenish TCA cycle:

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

185N

20NH3

Glutamate

Glutamate DH

Amino Acid ProductionGlutamate

• accumulation of glutamate and thus α-ketoglutarate removal

requires 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

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

185N

20NH3

Glutamate

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)