Download 06a Organic Acids 2

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
Acetic Acid and Vinegar Production
-4 0
12 2
20
10 2
20
82
ETP
6 ATP
00
12 2 = CH3-CH2OH
2 0 = 2 red. equiv.
10 2 = CH3-CH2O
-4 0 = O2
8 2 = CH3-COOH
Bacteria
Underoxidiser: Gluconobacter
Overoxidiser: Acetobacter (can totally oxidise to CO2)
Processes
Leave wine open to air
→ surface process
Trickling generator with wood
shavings
Submersed process (CSTR)
+ more economic
- Lower taste quality
Wood
Shavings
Acetic Acid and Vinegar Production
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%
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
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?
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
10 3
+ 0 1 → 10 4
Anaplerotic sequences to replenish reactions of TCA cycle
(usually for biosynthesis)
TCA Cycle – Electron and Carbon Flow
Citric acid synthesis during trophophase
10 4
12 4
12 4
14 4
Glucose
10 3
Pyruvate
82
Acetyl-CoA
Citrate synthase
18 6
glycolysis
OAA
Malate DH
Malate
Fumarase
Fumarate
Succinate DH
Succinate
24 6
Citrate
Aconitase
18 6
Isocitrate
16 5
Isocitrate DH
α-ketoglutarate
α-ketoglutarate DH
How can the cycle continue when citrate is excreted?
TCA Cycle – Metabolites
8 2 Acetyl-CoA
CH2-COOH
Citrate COH-COOH
18 6
CH2-COOH
α-ketoglutarate HOOC-CH2-CH2-CO-COOH 1-6-6-2-1
10 4 OAA HOOC-CO-CH2-COOH
16 5
12 4
Fumarate HOOC-CH=CH-COOH 1-5-5-1
14 4 Succinate HOOC-CH2-CH2-COOH 1-6-6-1
12 4 Malate HOOC-CH2-CHOH-COOH 1-6-4-1
10 3
Pyruvate CH3-CO-COOH
How can the cycle continue when citrate is excreted?
TCA Cycle – Citrate isomerisation
Citrate
CH2 - COOH
|
HOCOH -COOH
|
CH2 - COOH
CH2 - COOH
|
cis-Aconitate CH - COOH
||
HOCH - COOH
CH2 - COOH
|
Iso-Citrate CH - COOH
|
HOCH - COOH
TCA Cycle – Metabolites
82
Acetyl-CoA
10 4 OAA HOOC-CO-CH2-COOH 1-2-6-1
18 6
CH2-COOH
Citrate 1-6-3-1-6-1 COH-COOH
CH2-COOH
16 5 α-ketoglutarate HOOC-CH2-CH2-CO-COOH 1-6-6-2-1
12 4 Fumarate HOOC-CH=CH-COOH 1-5-5-1
14 4 Succinate HOOC-CH2-CH2-COOH 1-6-6-1
12 4 Malate HOOC-CH2-CHOH-COOH 1-6-4-1
10 3 Pyruvate CH3-CO-COOH 7-2-1
TCA Cycle – Electron and Carbon Flow
Citric acid synthesis during idiophase
Pyruvate
carboxylase
OAA
10 4
Malate
12 4
Fumarate 12 4
Succinate
14 4
Glucose
10 3
Pyruvate
82
Acetyl-CoA
Citrate synthase
18 6
18 6
glycolysis
01
24 6
Citrate
Isocitrate
16 5 α-ketoglutarate
10 3 + 0 1 +
82
→ 10 4
Pyruvate + CO2 + Acetyl-CoA → Citrate
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))?
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
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
82
+
42
→ 12 4
→ 10 4 +
20
Acetate + Glyoxylate → Malate → OAA + 2 NADH
•What is the reaction that forms glyoxylate ?
•Can you think what is the most oxidised organic ?
Citric Acid Synthesis With Lipids as the Substrate
Glyoxylate is derived from isocitrate lyase reaction:
(see glyoxylate cycle)
42
→ 12 4
+
Isocitrate → Succinate + Glyoxylate
18 6
How can the excretion of citrate be guaranteed when isocitrate
is 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 Flow
Citric acid synthesis during trophophase
82
OAA
Malate DH
Malate
Fumarase
Fumarate
Succinate DH
Succinate
10 4
12 4
12 4
14 4
Acetyl-CoA
Citrate synthase
18 6
Citrate
Aconitase
18 6
Isocitrate
16 5
Isocitrate DH
α-ketoglutarate
α-ketoglutarate DH
How can the cycle continue when citrate is excreted?
Citric Acid Synthesis With Lipids as the Substrate
Glyoxylate Formation from Isocitrate Lyase
82
OAA
10 4
Acetyl-CoA
Citrate synthase
18 6
18 6
14 4
Citrate
Aconitase
Isocitrate
Isocitrate
lyase
42
Glyoxylate
(CHO-COOH)
Citric Acid Synthesis With Lipids as the Substrate
Glyoxylate use to lead to OAA via malate
82
82
OAA
10 4
Malate
12 4
14 4
Acetyl-CoA
Citrate synthase
18 6
18 6
Citrate
Aconitase
Isocitrate
Isocitrate
lyase
42
Glyoxylate
(CHO-COOH)
How can the excretion of citrate be guaranteed when isocitrate
is necessary for citrate synthesis?
Citric Acid Synthesis With Lipids as the Substrate
(Glyoxylate Cycle)
82
82
OAA
10 4
Malate
12 4
Malate synthase
Fumarate 12 4
Succinate
14 4
Acetyl-CoA
Citrate synthase
18 6
18 6
Citrate
Aconitase
Isocitrate
Isocitrate
lyase
42
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.
Citric Acid Synthesis With Lipids as the Substrate
(Glyoxylate Cycle)
Glyoxylate cycle can produce citrate from acetate only:
3 82
→
18 6 + 6 0
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
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
Citric Acid Production Industrial Problems
•Possible reaction of oxalic acid production:
42
→
22
+ 20
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
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
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
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 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
24 6
22 6
→
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
Amino Acid Production
Glutamate
Glutamate and lysine are the most significant commercial
amino acids produced by bioprocesses.
Strong competition existing from: Lysine: 11%
Rest: 2%
• chemical synthesis
• extraction from animal protein
Glutamate is the only mass product
Glutamate: 87%
Use:
Food additive (“flavour enhancer”) Japan, China,…
Sold as mono-sodium-glutamate (MSG)
Has had bad reputation because of over use.
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:
Glutamate Production 1
10 4
Glucose
10 3
Pyruvate
82
Acetyl-CoA
Citrate synthase
12 4
12 4
14 4
α-ketoglutarate DH
glycolysis
OAA
Malate DH
Malate
Fumarase
Fumarate
Succinate DH
Succinate
24 6
18 6
Citrate
Aconitase
18 6
Isocitrate
16 5
Isocitrate DH
α-ketoglutarate
Glutamate DH
20
NH3
18 5 N
Glutamate
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
10 3
+ 2 0 + 0 1 → 12 4
With hydrocarbons as the substrate: glyoxylate cycle is
operable (refer to citric acid production)
Glutamate Production 1
OAA
Malate DH
Malate
Fumarase
Fumarate
Succinate DH
Succinate
10 4
Glucose
10 3
Pyruvate
82
Acetyl-CoA
Citrate synthase
12 4
12 4
14 4
α-ketoglutarate DH
glycolysis
01 20
24 6
18 6
Citrate
Aconitase
18 6
Isocitrate
16 5
Isocitrate DH
α-ketoglutarate
Glutamate DH
20
NH3
18 5 N
Glutamate
Glutamate Production 2
(Feedback inhibition)
Glucose + NH3 → Glutamate + CO2 + 6H
24 6 + N
18 5 N + 0 1 + 6 0
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
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)