PDC

1
Ethanolic Fermentation - Electron and carbon flow
-
glucose
ATP
ATP
PDC
PDC
EDH
EDH
glucose
ethanol
2 red. equiv.
pyruvate
Key enzymes PDC pyruvate decarboxylase EDH
Ethanol dehydrogenase
acetaldehyde
ethanol
2
Ethanolic Fermentation - Electron and carbon flow
-
OH H C H H C H H
O.S. -1 ? 5 electrons
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)

3
The Entner Doudoroff (KDPG) pathway of ethanolic
fermentation
Organism Zymonas mobilis (not examined)
glucose
gluconate
GAP
pyruvate
ATP
CO2.
acetaldehyde
ethanol
4
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

5
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

6
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

7
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?
• 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 lt5)
• Examples Milk, whole meal flour, vegetables,

8
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)

9
Homolactic Fermentation - Electron and carbon
flow -
ATP
ATP
LDH
LDH
lactate
glucose
LDH lactate dehydrogenase
2 red. equiv.
pyruvate
lactate
10
Homo-lactic Fermentation - Electron and carbon
flow -
O CH C H C H H C H H
O.S. 3 ? 1 electron
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
11

• 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
• Dont degrade proteins
• Dont 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).

12
Heterolactic Fermentation Phosphoketolase pathway
glucose
ribose
2 red. equiv.
pyruvate
ATP
lactate
ethanol
acetate
CO2.
Phosphoketolase pathway combination of
Pentosephosphate cycle and FBP pathway
13
Heterolactic Fermentation Phosphoketolase pathway
glucose
ribose
2 red. equiv.
pyruvate
ATP
lactate
ethanol
acetate
CO2.
Presence of oxygen ? lactate, acetate and CO2
production ? 1 additional ATP from acetokinase.
No ETP
14
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).
15
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
• drying of fodder ? hay (25),
• ensilaging (10) (2ATP out of 38)

16
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
17
Applications of Lactic Fermentation
Sauerkraut

• Similar to silage with following modifications
• 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
18
Applications of Lactic Fermentation
Brine Recycle
19
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
gt0.5
7) Wooden barrels or plastic tanks
20
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
21
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
22
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
23
Cheese Production
Milk
Homogenise
Add starter culture (S. cremoris, S. lactis, L.
bulgaricus, S. thermophilus
Pasteurise
Add Rennet
Curdling Stirring Settling
Yougurt (430)
Scolding Cooling Washing Salting
Heat treatment (600) Kneading
Whey
Quark Fromage frais (acidic paste)
Whey
Cottage cheese (granular)
Pressuring Maturing
Proteolytic enzyme Coagulating
Heated stirring
Brie Edamer
Cheddar
24
Propanoate Formation From Lactate

• Acryloyl pathway (Clostridium propionicum)
• The 4 reducing equivalents from lactate oxidation
  to acetate
• are merely dumped onto two further moles of
  lactate
• (dismutation, disproportionation)

LDH
PrDH
PDH
ATP
Enzymes Lactate DH, Pyruvate DH, Propionate DH
(PrDH)
25
Propanoate Formation From Lactate

1. Acryloyl pathway (Clostridium propionicum)

Energetic benefit? The excretion of acetate
gains 1 ATP (acetate kniase), Thus 1/3
ATP/lactate metabolised.
LDH
PrDH
PDH
ATP
How to generate ATP from acetate
excretion Phosphate Acetyl transferase AcetateCo
A Pi ? Acetyl-P CoA Acetokinase Acetyl-P
ADP ? Acetate ATP
26
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
27
Propanoate Formation From Lactate
2. Methyl-Malonyl-Pathway (Propionibacteria)
lactate
LDH
propionate
PDH
ATP
succinate
fumarate (malate)
ATP
OAA
pyruvate
28
Propionic Fermentation of Glucose
29
Propionic Fermentation of Glucose
30
Propionic Fermentation of Glucose
31
Butyric Fermentation
32
Acetone Butanol fermentation
33
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.
34
Homoacetogenesis