Biochemistry of Fermentation Processes

1. Biochemistry of Fermentation Processes David A. Boyles Professor of Chemistry Department of Chemistry and Chemical Engineering South Dakota School of Mines and Technology

2. I. Overview of Fermentation II. Biochemistry of Fermentation

3. Fermentation Background Known since antiquity Earliest use of term referred to natural fermentation by wild and unidentified microbes Distinguish two kinds • Indigenous Fermentations • Technological Fermentations

4. INDIGENOUS FERMENTATIONS Fermentation originally used to produce foods and beverages Many products have been standardized and commercialized Ales—natural yeastsCheeses—natural fungiWines—natural yeasts Many others are produced commercially in limited quantities for specialized markets, or remain uncommercialized and are products of indigenous, local cultures kefir, kim-chi, sauerkraut, yoghurt, San Francisco sourdough bread…

5. Advantages of Indigenous Products • Unique flavor profile Enhanced storage Disadvantages of Indigenous Products Quality control—natural variations over time, possibility of contamination Difficult to mass produce

6. Fermentation: Current Definitions In the strict biochemical sense of the term fermentation involves the action of anaerobic organisms on organic substrates Modern usage extends definition to the microbiological formation of smaller organic molecules, whether aerobic or anaerobic The component products of fermentation may be isolated from the feedstock and purveyed as pure substances, unlike fermentation of antiquity: eg., ethanol versus wine

7. Technological Fermentation: Features • Large scale reactors for commercial production • Carefully controlled conditions • Optimized yields of pure products • Pure strains of microbes • Genetically engineered microbes by recombinant technologies allowing production of rare natural products such as insulin, growth hormones, enzymes

8. Variety of Isolated Fermentation Products Classical Fermentation Products Before 1950 • Organic molecules of six or fewer carbons Current Fermentation Products • Amino acids, and even (loosely) includes proteins such as insulin, HGH, polysaccharides

9. Criteria for Potential Industrial Chemical Products and Transformations • Favorable demand eg., Citric acid • Reliable supply eg., petroleum, starch • Technological Knowledge eg., intellectual capital • Profitability eg., value added • Downstream Utilization eg., food additive • Merchandising eg., ‘THIS IS IT!’

10. Dateline • 1859 • Edwin Drake • Oil industry began in Titusville, Pennsylvania • 1865 • Louis Pasteur • 1865 process to inhibit fermentation of wine and milk • 1903 • Henry Ford founds Ford Motor Company in 1903 • Model T Automobile: By 1927, 15 million had been sold • 1910 to 1919 • WWI • 1939 to 1945 • WWII

11. Classic Fermentation Productsfrom Technology • Ethanol • Acetone and n-Butyl Alcohol • Organic Acids • Citric Acid • Acetic Acid • Lactic Acid • Itaconic Acid

12. Fermentation: Scale Production will never replace petroleum-based chemicals Not enough agricultural biomass available Biomass is oxygen-rich, unlike petroleum which is carbon-rich, reducing mass Production will serve to augment petroleum-based chemicals

13. Classic Fermentation Products I industrial solvent, beverage, fuel Saccharomyces cerevisiae solvent Clostridium acetobutylicum food and pharmaceutical use Lactobacillus delbrukki, bulgaricus synthetic rubber Bacillus polymyxa, Acetobacter aerogenes

14. Classic Fermentation ProductsII • Acetic Acid—Saccharomyces sp., Acetobacter • Lactic Acid—Lactobacillus delbruckii • Citric Acid—Aspergillus niger • Itaconic Acid—Aspergillus itaconicus

15. Ethanol C2 • 1906 in US Industrial Act—denatured product was legalized in the US • WWII: demands for industrial product increased—use for synthetic rubber and smokeless gunpowder • Whole grains, starches, sulfite liquors or saccharine materials are used as feed stocks • Saccharomyces cerevesiae cannot ferment starch directly—amylases must first break down starch to sugars

16. Organic Acids Vinegar C2 • French name vin + aigre • Condiment and preservative • Feedstock: sugary or starchy • Slow Process: Orleans or French method --”mother of vinegar” • Generator Process: 1670 --fast process, maximum air exposure • Cider (apples), wine (grapes), malt (barley), sugar, glucose, spirit (grain) used for biomass

17. Organic Acids Lactic Acid C3 • 1790 by Scheele from milk • Present in sour milk, sauerkraut, bread, muscle tissue, principal organic soil acid • 1881 Commercial production by Chas. Avery, Littleton, Mass as substitute for cream of tartar • Dextrose, maltose, lactose, sucrose, whey Starch, grapefruit, potatoes, molasses, beet juice • Dimerizes to lactide upon heating PURAC for applications

18. Glycerol C3 • Principal source is saponification of fats and oils • Diverse use in explosives, foods, beverages, cosmetics, plastics, paints, coatings • First identified by Pasteur • WWI demand exceeded supply, esp. in Germany—became leader in fermentation • At least one integrated plant took directly to nitroglycerine

19. Acetone-Butanol C3 and C4 • True, anaerobic fermentation by Clostridium • Major development during WWI: used for synthetic rubber via butadiene; critical commodity for cordite • WWII production was solely by fermentation • 1861 Pasteur first observed formation; 1905 Schardinger • 1916 Chaim Weizmann procedure first industrial use in Canada, Terre Haute for WWI production • 1926 Demand for lacquers: Peoria • 96 fermentors in use, cap. 50,000 gallons each

20. 2,3-Butanediol C4 • Major interest in WWII by US and Canada • Northern Regional Research Laboratory of USDA in Peoria • Uses as antifreeze, butadiene synthesis • 1936, Julius Nieuwland of Notre Dame with DuPont’s Wallace Carothers--DuPrene (neoprene) from it and later from petroleum sources • Fermentation sources never commercialized

21. Organic Acids Itaconic Acid C5 • Resin and detergent industries • Polymerizable alkene • Competition with methacrylate • Also produced by pyrolysis of citric acid • Commercial production since 1940s • Surface culture method—shallow pans • Submerged culture method—vats • Corn steep liquor: mixture of aa and sugars

22. Organic Acids Citric Acid C6 • Made today by mold fermentation • 1893: Carl Wehmer discovery • 1917: Currie surface fermentation method • 1945 Commercial, Landenburg Germany • Molasses, cane blackstrap molasses, sugar • Remarkable increase in production over past 60 years—huge sales to China • Originally produced directly from citrus fruit

23. Biochemistry of Fermentation A. Overall Strategy B. Bioenergetics • Energy transfer from highly negative DG to less negative DG • Harvesting of electrons • Temporary energy storage C. Major metabolic pathways and cycles

24. A. Overall Strategy • Organic molecules “contain” energy • True interest is twofold atoms electrons • Living organisms strip organic foodstuffs of electrons and successively oxidize foodstuffs in order to carry out life processes • Organic foodstuffs become successively more oxidized and may be released to atmosphere ultimately as CO2

25. B. Bioenergetics • Energy must be stored in temporary, highly available chemical form • Adenosine triphosphate is the universal energy storage molecule • Electrons must be transported by organic molecules in the form of utilizable “reducing equivalents” • Nicotinamide adenine dinucleotide and flavin adenine dinucleotide are the universal electron carriers

26. ATP • Energy of organic molecules is not useable to living organisms—requires conversion into the “currency” of the cell, ATP, adenosine triphosphate • ATP has an intermediate energy of hydrolysis • DG of hydrolysis is –7.3 kcal/mol • Low compared to some, high compared to other hydrolyses • ATP levels must be kept constant in all cells for life processes to continue to occur

27. Electron Carriers • Electrons stripped from foodstuffs must be transported • Two universal electron carriers are used • Nicotinamide adenine dinucleotide NAD • Flavin adenine dinucleotide FAD • Both are found in conjuction with enzymes, thus are termed “coenzymes”

28. NAD accepts two electrons and a proton (H+) to form NADH • FAD accepts two electrons and two protons to form FADH2 • Both NADH and FADH2 are termed “reducing equivalents” since they carry electrons

29. In Summary Have Three Players To Consider in ALL Metabolic Pathways • Energy carrier molecule • Electron carrier molecules • Organic compounds at various oxidation states along the way • Glucose to A to B to C to D to E to carbon dioxide

30. C. Major Metabolic Pathways and Cycles • Definition • Particular pathways and cycles

31. Metabolism: Definition and Types • Metabolism is a sequence of discrete chemical transformations (chemical reactions) • No reaction is at all foreign to organic chemistry • Two Kinds of Metabolism • Catabolic—complex organics to simpler • Anabolic—simpler organics to complex • Both operate simultaneously by different sequences of chemical transformations

32. Each reaction in the sequence requires a specific enzyme A B C • The linked sequence is a ‘pathway’ • Each enzyme is specific for its substrate • Regulation of the pathway is possible since some enzymes can be activated, and others inhibited E2 E1

33. Metabolism: Specific Pathways and Cycles • Glycolysis • Citric Acid Cycle • Electron Transport Chain

34. Glycolysis • Central pathway in most organisms • Embden-Meyerhof Pathway • Begins with glucose C6 • Requires 10 discrete steps • Ends with pyruvate 2 X C3 • Anaerobic pathway--primitive

35. Glycolysis: Features • Textbook, page 133 • One glucose is ‘split’ (glucose + lysis = glycolysis) • The splitting step is a reverse aldol condensation

36. Final pyruvate has several possible fates • Fates depend on • Organism • Conditions • Tissue • Conversion by • Decarboxylation to ethanol 2C and carbon dioxide 1C • Decarboxylation to Acetyl CoA 2C and carbon dioxide • Reduction by NADH to lactate 2C; regenerates NAD+

37. One Fate: Alcoholic Fermentation • Yeast ferment glucose to ethanol and carbon dioxide, rather than to lactate • Sequence: pyruvate acetaldehyde ethanol

38. Glycolysis: Summary Schematic from Pyruvate Onward Glucose 10 marvelous steps! 2 Pyruvate Anaerobic conditions Anaerobic conditions O2 -2CO2 2 Lactate 2 EtOH + 2 CO2 Some organisms, contracting muscle Alcoholic fermentation 2 Acetyl CoA O2 Citric Acid Cycle: Aerobic conditions—animal, plant, microbial cells 4CO2 and 4 H2O

39. Glycolysis Energetics • Standard Free Energy for calorimetric oxidation of glucose to carbon dioxide and water is –686 kcal/mol • Glycolytic degradation of glucose to two lactate (DG = -47.0 kcal/mole) (47/686) X 100 = 6.9 percent of the total energy that can be set free from glucose This does NOT mean anaerobic glycolysis is wasteful, but only incomplete to this point of metabolism!

40. Citric Acid Cycle • Background • Function • Schematic

41. TCA: Background • Kreb’s Cycle, Tricarboxylic Acid Cycle • Sir Hans Krebs 1930’s • Regarded as the most single important discovery in the history of metabolic biochemistry • Is a true cycle: not a linear pathway

42. TCA: Function • To continue to strip remaining energy from pyruvate on its way to carbon dioxide which is released to atmosphere • To produce organic molecules which may be drained off the cycle for anabolic purposes • To continue to harvest electrons from pyruvate • To serve as a central collecting pool for foodstuffs originating from molecules other than glucose

43. TCA: Schematic Pyruvate 3C Fatty acids Amino acids Acetyl CoA 2C Oxaloacetate 4C Citrate 6C Isocitrate Malate Note: Sequence is Clockwise +2 carbon dioxide Fumarate + NADH + FADH2 Alpha-ketoglutarate Succinyl CoA Succinate

44. Electron Transport Chain Organization of “Chain” Electron Carriers in Chain Electron Carriers: Free Energy Changes Direction of Flow via Electron Carriers Ultimate Fate of Electrons and Protons

45. ETC: Organization of “Chain” • The physical electron carriers are molecules embedded in the cell membrane as free-floating bodies See Figure 5.6 page 137 in your textbook • Likened to buoys that bob and move to carry electrons from one carrier to the other • Also often likened to a bucket brigade

46. ETC: Electron Carriers in Chain A ‘carrier’ both accepts and then donates electrons Thus, carriers undergo reversible oxidation and reduction Variety of electron carriers are used, eg. Flavoproteins Cytochromes—copper containing FeS Centers Coenzyme Q: a quinone

47. Electron Carriers: Free-Energy Changes Electrons flow from electronegative toward electropositive “carriers” This is the result of the loss of free energy, since electrons always move in such a direction that the free energy of the reacting system: DECREASES! The free energy decreases for spontaneous changes! Electrons move spontaneously from negative to more positive standard reduction potentials

48. Direction of Electron Flow via Electron Carriers 0 -0.4 NADH FMN 10 0.0 CoQ cyt b Eo’ 20 +0.2 kcal cyt c 30 Protons are pumped across membrane at each incremental drop +0.4 40 cyt a ?? +0.8 50 Direction of Electron Flow is Consistent with Thermodynamics

49. Direction of Electron Flow is Consistent with Thermodynamics Reduction Potentials measure the ‘natural’ (inherent) tendency of substances to gain electrons (be reduced) That is, oxygen has the most positive reduction potential of all electron acceptors in the chain The more positive the reduction potential, the more the substance wants to gain electrons Some substances “naturally” gain electons more easily than others: in the electron transport chain, oxygen gains them most easily of all Reduction potentials are easily related to free energy changes by the Faraday equation

50. ETC: Fate of Electrons Oxygen O2 is the ultimate electron and proton acceptor Since this is the only stage of metabolism at which oxygen (O2) is used, the electron transport chain is referred to as the • RESPIRATORY TRANSPORT CHAIN