1. 10 3 2 0 3 2 2 12 2 2 2 0 0 1 0 1 PDC EDH ethanol ATP 24 6 glucose 24 6 2 0 10 3 2 12 2 = glucose = 2 red. equiv. = pyruvate = acetaldehyde = ethanol Ethanolic Fermentation - Electron and carbon flow - Key enzymes: PDC = pyruvate decarboxylase EDH = Ethanol dehydrogenase

2. OH HCH 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

3. 22 6 2 0 10 2 3 3 12 2 3 2 0 0 1 0 1 ATP 24 6 6 0 1 10 3 2 12 2 = glucose = CO2. = pyruvate = acetaldehyde = ethanol 10 2 12 2 24 2 = gluconate 12 3 = GAP The Entner Doudoroff (KDPG) pathway of ethanolic fermentation Organism: Zymonas mobilis (not examined)

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? : 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,

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. 10 3 2 0 3 12 3 3 2 0 LDH lactate ATP 24 6 6 2 0 10 3 12 3 = glucose = 2 red. equiv. = pyruvate = lactate Homolactic Fermentation - Electron and carbon flow - LDH = lactate dehydrogenase

10. OCH C HCH 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

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 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).

12. 20 5 2 0 10 3 12 3 2 0 ATP 24 6 6 0 1 10 3 12 2 = glucose = CO2. = pyruvate =acetate = ethanol 12 2 20 5 = ribose 12 3 = lactate 8 2 2 0 0 1 2 0 = 2 red. equiv. 8 2 Heterolactic Fermentation Phosphoketolase pathway Phosphoketolase pathway = combination of Pentosephosphate cycle and FBP pathway

13. 20 5 2 0 10 3 12 3 2 0 ATP 24 6 6 0 1 10 3 12 2 = glucose = CO2. = pyruvate =acetate = ethanol 12 2 20 5 = ribose 12 3 = lactate 8 2 2 0 0 1 2 0 = 2 red. equiv. 8 2 Heterolactic Fermentation Phosphoketolase pathway 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: 1.drying of fodder  hay (25%), 2.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: 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

18. Applications of Lactic Fermentation

19. 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

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 Pasteurise Add Rennet* Add starter culture (S. cremoris, S. lactis, L. bulgaricus, S. thermophilus Yougurt (430°) Curdling** Stirring Settling Heat treatment (600°) Kneading Whey Scolding*** Cooling Washing Salting Whey Quark Fromage frais (acidic paste) Cottage cheese (granular) Pressuring Maturing Brie Edamer Cheddar * Proteolytic enzyme ** Coagulating *** Heated stirring

24. 2 0 12 3 2 0 ATP 14 3 12 3 3 14 3 8 2 0 1 LDH PDH 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) Enzymes: Lactate DH, Pyruvate DH, Propionate DH (PrDH) PrDH

25. 2 0 12 3 2 0 ATP 14 3 12 3 3 14 3 8 2 0 1 LDH PDH Propanoate Formation From Lactate 1.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 excretion Phosphate Acetyl transferase: Acetate~CoA + 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. 2 0 14 4 2 0 ATP 14 3 12 3 3 14 3 8 2 0 1 LDH PDH 12 4 10 4 3 12 3 ATP Fd ETC Vit B12 0 1 2 0 12 3 10 3 4 = lactate = pyruvate = OAA 14 3 = propionate 12 4 = fumarate (malate) 14 4 = succinate Propanoate Formation From Lactate 2. Methyl-Malonyl-Pathway (Propionibacteria)

28. 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