103 20 103

102 102

122 122

20

0101

PDCPDC

EDH EDH

ethanol

ATPATP

246glucose

246

20

103

102

122

= glucose

= 2 red. equiv.

= pyruvate

= acetaldehyde

= ethanol

Ethanolic Fermentation- Electron and carbon flow -

Key enzymes:PDC = pyruvate decarboxylaseEDH = Ethanol dehydrogenase

OH

H C H

H C H

H

Ethanolic Fermentation- Electron and carbon flow -

• Energy conserved: 2 ATP from glycolysis (PGK, PK)• Key enzymes: •Pyruvate Decarboxylase, •Ethanol Dehydrogenase

(could also be called ethanol oxidase or acetaldehyde reductase)

O.S.: -1 → 5 electrons

O.S.: -3 → 7 electrons

226

20

102

103 103

122

123

20

0101

ATP

246 246

01

103

102

122

= glucose

= CO2.

= pyruvate

= acetaldehyde

= ethanol

102

122

242 = gluconate

123 = GAP

The Entner Doudoroff (KDPG) pathway of ethanolic fermentationOrganism: Zymonas mobilis(not examined)

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 degradation2) 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 strains5) 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)

103 20 103

123 123

20

LDH LDH

lactate

ATPATP

246

246

20

103

123

= glucose

= 2 red. equiv.

= pyruvate

= lactate

Homolactic Fermentation- Electron and carbon flow -

LDH = lactate dehydrogenase

O CH

C

H C H

H C H

H

Homo-lactic Fermentation- Electron and carbon flow -

O.S.: 0 → 4 electrons

O.S.: -3 → 7 electrons

O.S.: +3 → 1 electron

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).

•

205

20

103

123

20

ATP

246 246

01

103

122

= glucose

= CO2.

= pyruvate

=acetate

= ethanol

122

205= ribose

123= lactate

8220

01 20= 2 red. equiv.

82

Heterolactic FermentationPhosphoketolase pathway

Phosphoketolase pathway = combination of Pentosephosphate cycle and FBP pathway

205

20

103

123

20

ATP

246 246

01

103

122

= glucose

= CO2.

= pyruvate

=acetate

= ethanol

122

205= ribose

123= lactate

8220

01 20= 2 red. equiv.

82

Heterolactic FermentationPhosphoketolase pathway

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 materialProcess:

1) partial drying of fodder2) shredding3) Rapid filling of silo (1 or 2 days)4) packing as densely as possible5) Compressing6) Sealing airtight7) 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 FermentationSauerkraut

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 FermentationSauerkraut

Similar to silage with following modifications:1) White cabbage as the only plant material2) Cabbage mixed with NaCl (2 – 2.5%)3) Capacity of vessels (concrete, wood) up to

100 tons4) Incubation (18oC to 20oC) for 4 weeks5) Recirculation of brine by pumping for

process monitoring (acids)6) About 1.5% lactic acid produced7) 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

Brin

e R

ecyc

le

Brin

e R

ecyc

le

Applications of Lactic Fermentation

Applications of Lactic FermentationOlives

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 FermentationTechnical 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

HomogenisePasteurise

Add Rennet*Add starter culture(S. cremoris, S. lactis,L. bulgaricus,S. thermophilusYougurt (430°)

Curdling**StirringSettling

Heat treatment(600°)Kneading Whey

Scolding***CoolingWashingSalting

WheyQuarkFromage frais(acidic paste)

Cottage cheese(granular)

PressuringMaturing

BrieEdamer

Cheddar

* Proteolytic enzyme** Coagulating*** Heated stirring

20

123

20ATP

143

123 123

143

82

01

LDH

PDH

Propanoate Formation From Lactate1. Acryloyl pathway (Clostridium propionicum)

The 4 reducing equivalents from lactate oxidation to acetateare merely “dumped” onto two further moles of lactate(dismutation, disproportionation)

Enzymes: Lactate DH, Pyruvate DH, Propionate DH (PrDH)

PrDH

20

123

20ATP

143

123 123

143

82

01

LDH

PDH

Propanoate Formation From Lactate1. Acryloyl pathway (Clostridium propionicum)

PrDH

Energetic benefit?

The excretion of acetate gains 1 ATP (acetate kniase),

Thus 1/3 ATP/lactate metabolised.

How to generate ATP from acetate excretionPhosphate Acetyl transferase:Acetate~CoA + Pi → Acetyl-P + CoAAcetokinase:Acetyl-P + ADP → Acetate + ATP

Propanoate Formation From Lactate

2. Methyl-Malonyl-Pathway (Propionibacteria)

• 2 reducing equivalents from lactate oxidation (exactly: PDHand ferredoxin as e- carrier) are transferred via electrontransport 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 respirationHomoacetogenesis is another example

20

144

20ATP

143

123 123

143

82

01

LDH

PDH

124

104

103

123

ATP

Fd

ETC

Vit B12

01

20

123

103

104

= lactate

= pyruvate

= OAA

143= propionate

124 = fumarate (malate)

144= succinate

Propanoate Formation From Lactate2. Methyl-Malonyl-Pathway (Propionibacteria)

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