Transcript 05 Fermentations 2008

Ethanolic Fermentation
- Electron and carbon flow 24 6 glucose
ATP
ATP
01
10 3
PDC
20
20
10 2
01
10 2
EDH
12 2
10 3
PDC
EDH
ethanol
24 6 = glucose
12 2
20
= 2 red. equiv.
10 3 = pyruvate
Key enzymes:
PDC = pyruvate decarboxylase
EDH = Ethanol dehydrogenase
10 2 = acetaldehyde
12 2 = ethanol
Ethanolic Fermentation
- Electron and carbon flow OH
O.S.: -1 → 5 electrons
H
C
H
H
C
H
H
O.S.: -3 → 7 electrons
• Energy conserved:
2 ATP from glycolysis (PGK, PK)
• Key enzymes:
•Pyruvate Decarboxylase,
•Ethanol Dehydrogenase
(could also be called ethanol oxidase or acetaldehyde reductase)
The Entner Doudoroff (KDPG) pathway of ethanolic fermentation
Organism: Zymonas mobilis
(not examined)
24 6
24 6 = glucose
20
22 6
24 2 = gluconate
12 3 = GAP
12 3
20
10 3
10 3
10 2
10 2
12 2
12 2
10 3 = pyruvate
ATP
01
01
01
= CO2.
10 2 = acetaldehyde
12 2
= ethanol
Special features of Entner Doudoroff pathway
• 1 NADH, 1 NADPH
• Only 1 ATP (less biomass as byproduct)
• Only one pyruvate through GAP (bottleneck) → faster?
Special features of Zymomoanas
• Higher glucose tolerance
• Higher product yield (less ATP → less biomass) (100 g
ethanol / 250 g glucose) = 78% molar conv. eff
• Not higher ethanol tolerance
Special features of Entner Doudoroff pathway (not examined)
• 1 NADH, 1 NADPH
• Only 1 ATP (less biomass as byproduct)
• Only one pyruvate through GAP (bottleneck) → faster?
Special features of Zymomoanas
• Higher glucose tolerance
• Higher product yield (less ATP → less biomass) (100 g
ethanol / 250 g glucose) = 78% molar conv. eff
• Not higher ethanol tolerance
Ethanol as fuel in Brasil
• Distillation costs more energy than ethanol fuel value
• Separation costs higher than fermentation costs
Research
• Thermophilic strains (Clostridium using cellulose)
• Finding more ethanol resistant strains
Lactic Fermentation - Occurrence If plant or animal material containing sugars and complex nitrogen
sources is left in the absence of oxygen → lactic acid bacteria take
over 
Selective enrichment
Natural fermentation (since prehistoric times)
Why do lactic acid bacteria take over sugar conversion on rich
media? :
1) Simple metabolism → fast degradation
2) Amino acids are not synthesized but taken up from the medium →
faster growth
3) Strains are existing on substrate (e.g. milk, vegetables)
4) O2 tolerance of strains
5) Production of inhibitory acid (ph <5)
Examples: Milk, whole meal flour, vegetables,
Lactic Fermentation - Organisms Lactic acid bacteria (Lactobateriacease)
• gram positive
• non motile
• obligate anaerobics
• no spores
• aerotolerant
• no cytochromes and catalase
• fermentation of lactose
• no growth on minimal glucose media
• requirement of nutritional supplements (vitamins, amino acids, etc.)
• when supplied with porphyrins → they form cytochromes !?!
(indicating that they were originally aerobic organisms that have lost
the capacity of respiration, metabolic cripples)
Homolactic Fermentation
- Electron and carbon flow 24 6
ATP
ATP
10 3
20
LDH
12 3
20
10 3
LDH
lactate
12 3
24 6 = glucose
LDH = lactate dehydrogenase
20
= 2 red. equiv.
10 3 = pyruvate
12 3 = lactate
Homo-lactic Fermentation
- Electron and carbon flow O
CH
C
O.S.: +3 → 1 electron
H
C
H
H
C
H
H
O.S.: 0 → 4 electrons
O.S.: -3 → 7 electrons
Strategy:
1) Aerotolerant → can ferment with strict anaerobes are
still inhibited by oxygen
2) Simple quick metabolism and usage of carbohydrates
3) Production of acid, inhibiting competitors
Significance:
Why do lactic acid bacteria not spoil food but preserve it?
•Only ferment sugars (24 e-) to lactate (2* 12 e-)  nutritional value not
significantly altered
•Don’t degrade proteins
•Don’t degrade fats
•Acidity suppresses growth of food spoiling organisms (eg. Clostridia)
•enhances nutritional value of organic material (example sauerkraut, Vit. C,
scurvy)
• Complex flavour development (diacetyl)
•Examples:
•Yogurt, sauerkraut, buttermilk, soy sauce, sour cream, cheese, pickled
vegetables,
•technical lactic acid for the production of bio-plastic (hydroxy acids allow
chain linkages via ester bonds between hydroxy and carboxy group).
•
Heterolactic Fermentation
Phosphoketolase pathway
24 6
20
20
01
20 5
ATP
24 6 = glucose
20 5 = ribose
2 0 = 2 red. equiv.
10 3 = pyruvate
12 3 = lactate
10 3
20
82
12 2 = ethanol
8 2 =acetate
12 3
12 2
Phosphoketolase pathway = combination of
Pentosephosphate cycle and FBP pathway
0 1 = CO2.
Heterolactic Fermentation
Phosphoketolase pathway
24 6
20
01
20 5
ATP
20
24 6 = glucose
20 5 = ribose
2 0 = 2 red. equiv.
10 3 = pyruvate
12 3 = lactate
10 3
20
82
12 2 = ethanol
8 2 =acetate
12 3
12 2
0 1 = CO2.
Presence of oxygen → lactate, acetate and CO2 production
→ 1 additional ATP from acetokinase. No ETP
Heterolactic Fermentation
Organisms: E.g. Leuconostoc spp. Lactobacillus brevis
Strategy:
• Use of parts of the pentose phosphate cycle which is
designed for synthesis of pentose (DNA, RNA). →
• Aerotolerant, simple pathway, quick metabolism, suited for
substrate saturation.
Application: Sourdough bread, Silage, Kefir, Sauerkraut,
Gauda cheese (eyes)
In the presence of oxygen, reducing equivalents from glucose
oxidation are transferred to oxygen, allowing the gain of an
additional ATP via acetate excretion
Key enzymes of FBP pathway missing (Aldolase,
Triosephosphate isomerase).
Application of Lactic Fermentation
Silage: Lactic acid fermentation of fodder material
Process:
1) partial drying of fodder
2) shredding
3) Rapid filling of silo (1 or 2 days)
4) packing as densely as possible
5) Compressing
6) Sealing airtight
7) Additives (germination inhibitors, sugars, organic acids)
8) Avoid contamination with decaying fodder (Clostridia,
proteolytic bacteria)
Nutrient loss:
1. drying of fodder  hay (25%),
2. ensilaging (10%) (2ATP out of 38)
Applications of Lactic Fermentation
Sauerkraut
In principle identical to silage with following modifications:
1) White cabbage as the only plant material
2) Cabbage mixed with NaCl (2 – 2.5%)
3) Capacity of vessels (concrete, wood) up to 100 tons
4) Incubation (18oC to 20oC) for 4 weeks
5) Recirculation of brine by pumping for process monitoring
(acids)
6) About 1.5% lactic acid produced
7) Sterilisation of product to have cooked sauerkraut (German).
Raw (fresh sauerkraut used in salads)
8) Problem: 1 to 15 tons of highly polluted effluent per ton of
cabbage
Applications of Lactic Fermentation
Similar to silage with following modifications:
1) White cabbage as the only plant material
2) Cabbage mixed with NaCl (2 – 2.5%)
3) Capacity of vessels (concrete, wood) up to
100 tons
4) Incubation (18oC to 20oC) for 4 weeks
5) Recirculation of brine by pumping for
process monitoring (acids)
6) About 1.5% lactic acid produced
7) Sterilisation of product to have cooked
sauerkraut (German). Raw (fresh
sauerkraut used in salads)
8) Problem: 1 to 15 tons of highly polluted
effluent per ton of cabbage
Brine Recycle
Sauerkraut
Brine Recycle
Applications of Lactic Fermentation
Applications of Lactic Fermentation
Olives
1) Black (ripe) or green (unripe) olives
2) Pretreatment with 1.5% NaOH saline (reducing bitterness)
3) Washing
4) Place fruit (still alcaline) in brime of 10% NaCl + 3%
lactic acid (to neutralise pH)
5) Sugar addition to accelerate fermentation (Lactobacillus
plantarum)
6) Incubate for several months until lactic acid >0.5%
7) Wooden barrels or plastic tanks
Pickled Gherkins
1. Cover gherkins in 3% salt brine (NaCl)
2. Add spices, herbs, dill
3. Irradiate surface (UV) and close vessel
4. After 3 – 6 weeks 3% lactic acid is produced
5. Fermentation pattern like silage
Applications of Lactic Fermentation
Technical lactic acid
Use: Leather – Textile – and Pharmaceutical Industry
Bioplastics (Polylactic acid, biodegradable)
Food acid (flavourless, non volatile) e.g. in sausages
Product yield: 900 g per g of sugar
Substrate: whey, cornsteep liquor, malt extract,
ideally: sugars (15% cane or beets)
Strains: Lactobacillus bulgaricus, Lactobacillus delbrueckii
Duration: 5 days batch culture
Applications of Lactic Fermentation
Sourdough bread
Biological raising agent (homo- and heterolactic fermentation)
CO2 produced from heterolactic bacteria
Necessary for rye bread to increase digestibility
Health bread (lipid, proteins unchanged, vitamins produced)
Pre-acidified (stomach friendly)
Complex flavour development
Increased shelf life
Cheese Production
Milk
Homogenise
Pasteurise
Add Rennet*
Yougurt (430°)
Heat treatment
(600°)
Kneading
Quark
Fromage frais
(acidic paste)
* Proteolytic enzyme
** Coagulating
Brie
*** Heated stirring
Edamer
Curdling**
Stirring
Settling
Whey
Whey
Pressuring
Maturing
Add starter culture
(S. cremoris, S. lactis,
L. bulgaricus,
S. thermophilus
Scolding***
Cooling
Washing
Salting
Cottage cheese
(granular)
Cheddar
Propanoate Formation From Lactate
1. Acryloyl pathway (Clostridium propionicum)
The 4 reducing equivalents from lactate oxidation to acetate
are merely “dumped” onto two further moles of lactate
(dismutation, disproportionation)
12 3
LDH
12 3
20
PDH
PrDH
20
01
14 3
14 3
ATP
82
12 3
Enzymes: Lactate DH, Pyruvate DH, Propionate DH (PrDH)
Propanoate Formation From Lactate
1. Acryloyl pathway (Clostridium propionicum)
12 3
LDH
12 3
20
PDH
PrDH
The excretion of acetate gains 1
14 3 ATP (acetate kniase),
14 3
20
01
Energetic benefit?
Thus 1/3 ATP/lactate metabolised.
ATP
82
12 3
How to generate ATP from acetate excretion
Phosphate Acetyl transferase:
Acetate~CoA + Pi → Acetyl-P + CoA
Acetokinase:
Acetyl-P + ADP → Acetate + ATP
Propanoate Formation From Lactate
2. Methyl-Malonyl-Pathway (Propionibacteria)
• 2 reducing equivalents from lactate oxidation (exactly: PDH
and ferredoxin as e- carrier) are transferred via electron
transport phosphorylation to fumarate (fumarate respiration)
resulting in one extra ATP (2/3 ATP/lactate metabolised).
• Reverse TCA cycle.
Fumarate reduction is an example of anaerobic respiration
Homoacetogenesis is another example
Propanoate Formation From Lactate
2. Methyl-Malonyl-Pathway (Propionibacteria)
12 3
12 3
12 3
LDH
20
14 3
14 3
PDH
14
20
3
01
Vit B12
Fd
ATP
14 4
82
ETC 14 4 0
1
12 4
ATP
12 4
20
10 4
10 3
12 3
= lactate
= propionate
= succinate
= fumarate
(malate)
10 4
= OAA
10 3
= pyruvate
Propionic Fermentation of Glucose
Propionic Fermentation of Glucose
Propionic Fermentation of Glucose
Butyric Fermentation
Acetone Butanol fermentation
Homoacetogenesis
The homoacetogenesis starts like the butyric acid fermentation:
1) Use of the fructose bisphosphate pathway (FBP) leading to 2 puruvate and 2
NADH.
2) Oxidative decarboxylation of pyruvate to acetyl-CoA, hydrogen gas and CO2.
3) In contrast to the butyric fermentation no acetoacetyl-CoA is formed. Instead two
acetyl-CoA are intermediate products.
Homoacetogenesis