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
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
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Acetic Acid and Vinegar Production
Downstream
Only filtering to remove biomass
Critical process conditions
30C (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
Gadens fermentation type II
Trophophase growth and complete substrate
oxidation to CO2
Idiophase deregulated TCA cycle due to iron
limitation
??a-ketoglutarate DH, ? Aconitase ? Isocytrate
lyase, ? Citrate synthase. Why?
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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)
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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
a-ketoglutarate
Succinate
a-ketoglutarate DH
How can the cycle continue when citrate is
excreted?
9
TCA Cycle Metabolites
Acetyl-CoA
CH2-COOH Citrate COH-COOH
CH2-COOH
OAA HOOC-CO-CH2-COOH
a-ketoglutarate HOOC-CH2-CH2-CO-COOH 1-6-6-2-1
Fumarate HOOC-CHCH-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?
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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
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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
a-ketoglutarate HOOC-CH2-CH2-CO-COOH 1-6-6-2-1
Fumarate HOOC-CHCH-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
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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
a-ketoglutarate
Succinate
?


Pyruvate CO2 Acetyl-CoA ? Citrate
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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))?
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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-gtC3)
• ?plant seedlings, microbes, but not animals

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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 ?

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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)

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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
a-ketoglutarate
Succinate
a-ketoglutarate DH
How can the cycle continue when citrate is
excreted?
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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)
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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?
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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.
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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
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Citric Acid Production Industrial Problems

• Citrate is not a primary metabolite
• Not formed during exponential growth
• but under Fe limitation
• Continuous chemostat culture not suitable
• unless as multitank system
• ? Na ? yellow pigment and oxalic acid production
• ? Fe3 ? ? citric acid, ? oxalic acid, CO2
• ?No 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

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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 isocitrate lyase
for good reason If PEP is there then there is no
need to run glyoxylate cycle
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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
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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
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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 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
?
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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
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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.
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Amino Acid Production
Glutamate
Biochemistry Glycolysis, TCA cycle reductive
amination of a-ketoglutarate (glutamate DH)
block a-ketoglutarate DH accumulation of
a-ketoglutarate under excess of NH3 ?
glutamate accumulation accumulation of
glutamate and thus a-ketoglutarate
removal requires an anaplerotic sequence to
replenish TCA cycle
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Glutamate Production 1
Glucose
glycolysis
Pyruvate
Acetyl-CoA
Citrate
OAA
Citrate synthase
Malate DH
Aconitase
Malate
Isocitrate
Fumarase
Fumarate
Isocitrate DH
Succinate DH
a-ketoglutarate
Succinate
a-ketoglutarate DH
Glutamate DH
NH3
Glutamate
N
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Amino Acid Production
Glutamate
accumulation of glutamate and thus
a-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)
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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
a-ketoglutarate
Succinate
a-ketoglutarate DH
Glutamate DH
NH3
Glutamate
N
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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
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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 gt( 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)