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Biochemistry of Fermentation Processes David A. Boyles Professor of Chemistry Department of Chemistry and Chemical Engineering South Dakota School of Mines and Technology I. Overview of Fermentation II. Biochemistry of Fermentation 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 INDIGENOUS FERMENTATIONS Fermentation originally used to produce foods and beverages Many products have been standardized and commercialized Ales—natural yeasts Cheeses—natural fungi Wines—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… 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 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 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 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 Criteria for Potential Industrial Chemical Products and Transformations Favorable demand Reliable supply eg., Citric acid eg., petroleum, starch Technological Knowledge eg., intellectual capital Profitability eg., value added Downstream Utilization eg., food additive eg., ‘THIS IS IT!’ Merchandising 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 Classic Fermentation Products from Technology Ethanol Acetone and n-Butyl Alcohol Organic Acids – Citric Acid – Acetic Acid – Lactic Acid – Itaconic Acid 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 petroleumbased chemicals Classic Fermentation Products I Ethanol industrial solvent, beverage, fuel Saccharomyces cerevisiae Glycerol food and pharmaceutical use Lactobacillus delbrukki, bulgaricus Acetone-Butanol solvent Clostridium acetobutylicum 2,3-Butanediol synthetic rubber Bacillus polymyxa, Acetobacter aerogenes Classic Fermentation Products II Organic Acids Acetic Acid—Saccharomyces sp., Acetobacter Lactic Acid—Lactobacillus delbruckii Citric Acid—Aspergillus niger Itaconic Acid—Aspergillus itaconicus 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 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 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 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 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 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 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 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 Biochemistry of Fermentation A. B. Overall Strategy Bioenergetics – Energy transfer from highly negative DG to less negative DG – Harvesting of electrons – Temporary energy storage C. Major metabolic pathways and cycles 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 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 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 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” 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 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 C. Major Metabolic Pathways and Cycles Definition Particular pathways and cycles 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 A Each reaction in the sequence requires a specific enzyme E1 E2 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 Metabolism: Specific Pathways and Cycles Glycolysis Citric Acid Cycle Electron Transport Chain 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 Glycolysis: Features Textbook, page 133 One glucose is ‘split’ (glucose + lysis = glycolysis) The splitting step is a reverse aldol condensation 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+ One Fate: Alcoholic Fermentation Yeast ferment glucose to ethanol and carbon dioxide, rather than to lactate Sequence: pyruvate acetaldehyde ethanol Glycolysis: Summary Schematic from Pyruvate Onward Glucose 10 marvelous steps! Anaerobic conditions 2 Pyruvate O2 2 EtOH + 2 CO2 Alcoholic fermentation -2CO2 2 Acetyl CoA Anaerobic conditions 2 Lactate Some organisms, contracting muscle O2 Citric Acid Cycle: Aerobic conditions—animal, plant, microbial cells 4CO2 and 4 H2O 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! Citric Acid Cycle Background Function Schematic 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 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 TCA: Schematic Pyruvate 3C Amino acids Fatty acids Acetyl CoA 2C Oxaloacetate 4C Citrate 6C Isocitrate Malate Note: Sequence is Clockwise + NADH Fumarate + FADH2 Succinate +2 carbon dioxi Alpha-ketoglutarate Succinyl CoA 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 ETC: Organization of “Chain” The physical electron carriers are molecules embedded in the cell membrane as freefloating 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 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 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 Direction of Electron Flow via Electron Carriers -0.4 0.0 NADH 0 FMN 10 CoQ cyt b Eo’ 20 +0.2 +0.4 +0.8 kcal 30 cyt c Protons are pumped across membrane at each incremental drop 40 cyt a ?? 50 Direction of Electron Flow is Consistent with Thermodynamics Direction of Electron Flow is Consistent with Thermodynamics Reduction Potentials measure the ‘natural’ (inherent) tendency of substances to gain electrons (be reduced) Some substances “naturally” gain electons more easily than others: in the electron transport chain, oxygen gains them most easily of all 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 Reduction potentials are easily related to free energy changes by the Faraday equation 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 Synthesis of ATP Proton Pumping During ETC Processes Gradient Released via ATPase ATP Bookkeeping ATP Synthesis: Proton Pumping During Course of ETC As electrons are passed from one carrier to another along the chain, protons are pumped to the OUTSIDE of the membrane Protons build up outside the membrane, lowering pH A chemical gradient is thus produced ATP Synthesis: Gradient Released via ATPase The proton gradient formed during the electron transport chain is used to do work The protons are pumped back through an enzyme in the membrane, a process which catalyzes the formation of ATP (This concept of proton gradient used to do work is known as Peter Mitchell’s ‘chemiosmotic hypothesis’) This constitutes THE mechanism by which ATP is continuously provided for the steady-state storage of utilizable energy OXIDATIVE The process is known as PHOSPHORYLATION ATP Bookkeeping Each NADH molecule produced in any pathway is ultimately responsible for the production of 3 ATP Each FADH2 molecule produced is ultimately responsible for the production of 2 molecules of ATP nb: These ratios of 1:3 and 1:2 vary depending on organism (cf. page 137) ETC: Balance Sheet per Glucose Molecule Start to Finish Metabolic Stage NADH FADH2 Substrate Level Phos. Total ATP Glycolysis 0 produced = 0 ATP 2 = 2 ATP 2 ATP 6 ATP Pyruvate to Acetyl CoA 2 produced = 6 ATP 0 0 ATP 6 ATP 2 = 2 ATP 2 ATP (GTP) 24 ATP Kreb’s Cycle 6 produced = 18 ATP Cf. Table 5.1 page 138 Textbook Total 36 ATP Overall Energetics 36 ATP produced upon complete oxidation of glucose Multiplied times -7.3 kcal/mol per each ATP (energy of hydrolysis of ATP to ADP and inorganic phosphate) EQUALS TOTAL STORAGE OF 263 kcal ENERGY FROM GLUCOSE (263 kcal/686 kcal)/100 = 38% of energy in glucose conserved as ATP SUMMARY 1. The function of metabolism is to ensure the life of the organism 2. Oxidative pathways—first glycolysis, then the Kreb’s cycle—use electron carriers to harvest electrons 3. The electrons are passed through the electron transport chain, leading to a proton gradient 4. The proton gradient is used to do work by converting gradient energy to chemical energy in the form of high-energy ATP FINALLY Additional Pathways I Pentose-Phosphate Pathway –Serves to harvest electrons –Is an alternative glucose pathway –Produces 5C sugar intermediates critical for DNA and RNA synthesis (anabolism) These are referred to as purines in textbook, pg. 139 Figure 5.7 Additional Pathways II Amino Acid Anabolism: From TCA intermedicates Amino acids must be supplied for the growth requirements of all cells Example: Oxaloacetate to form glutamate Chemically, this is the reductive amination of a ketone to produce an amine