Lecture 10: Fermentation - BIOL371 Microbiology PDF

BIOL371: Microbiology Lecture 10 – Fermentation 1 Topics of today 1. 2. 3. 4. 5. 6. 7. Energetic and redox considerations Lactic and mixed acid fermentation Fermentations of obligate anaerobes Secondary fermentations Fermentations that lack substrate-level phosphorylation Syntrophy Hydrocarbon...

BIOL371: Microbiology Lecture 10 – Fermentation 1 Topics of today 1. 2. 3. 4. 5. 6. 7. Energetic and redox considerations Lactic and mixed acid fermentation Fermentations of obligate anaerobes Secondary fermentations Fermentations that lack substrate-level phosphorylation Syntrophy Hydrocarbon metabolism Materials covered:  Chapters 14.17-14.23  Figures 14.43-14.48, 14.50, 14.51, 14.53  Table 14.5, 14.6 2 Fermentations  Fermentations: Energy conservation depends primarily on substrate-level phosphorylation  Defined by lack of external electron acceptor  Achieve redox balance by donating electrons to metabolic intermediates excreted as fermentation products  Two major challenges  Conserve much less energy than respiratory organisms  Difficult to achieve redox balance  Tremendous reaction diversity  Many only ferment when lacking external electron acceptors anoxically  Many more exclusively fermentative 3 Energy-rich compounds and substrate-level phosphorylation  Energy-rich compounds contain an energy-rich phosphate bond or coenzyme A  These compounds are formed during fermentation  Allows microbe to make ATP by transferring the phosphate bond to ADP by substrate-level phosphorylation  Production of fatty acids is common in fermentations, gives opportunity to do substrate-level phosphorylation by producing fatty acid coenzyme-A derivative 4 Energy-rich compounds that can couple to substrate-level phosphorylation 5 Achieving redox balance  Total number of each type of atom and electrons in reactants/substrates and products must balance  Redox balance achieved by excretion of fermentation products; e.g., acids, alcohols  Redox balance often facilitated by H2 production  H2 is a powerful electron donor for respiration 6 Mechanisms of reducing protons to H2 during fermentation 7 Common fermentations and example organisms  Fermentations are classified by either the substrate fermented or the products formed (e.g., alcohol) 8 Homofermentative lactic acid bacteria  Lactic acid bacteria are Gram-positive nonsporulating bacteria that produce lactic acid as sole or major fermentation product  Homofermentative yields only lactic acid and two ATP/ glucose  Difference is the presence of aldolase from glycolysis 9 Heterofermentative lactic acid bacteria  Heterofermentation yields lactic acid, ethanol, CO2, and one ATP/glucose  Absence of aldolase from glycolysis  Observation of CO2 in culture distinguishes heterofermenters from homofermenters 10 Mixed-acid fermentations and butanediol production  Mixed-acid fermentations: characteristics of enteric bacteria; e.g., E. coli  Generate acetic, lactic, and succinic acids  Also typically formed are ethanol, CO2, and H2  Glycolysis used  Some produce neutral products such as butanediol 11 Obligate anaerobic fermenters  Many fermenters are obligate anaerobes  Grow under highly reducing conditions  Cannot tolerate O2, often produce H2 from fermentation • Clostridium species are obligately fermentative anaerobes • Ferment sugars, amino acids, purines and pyrimidines, and other compounds • Most ATP synthesis from substrate-level phosphorylation • Some generate proton motive force 12 Sugar fermentation by Clostridium  Saccharolytic (sugar fermenting); e.g., Clostridium pasteruianum  Produce butyric acid and H2 as major products  1.5 glucose converted to 3 pyruvates and 3 NADHs via glycolysis  Pyruvate from glycolysis split to acetyl Co-A, CO2, and ferrodoxinred  Cytoplasmic hydrogenase reduces H+ to H2 to balance redox 13 Amino acid fermentation and the Stickland reaction  Amino Acid Fermentation by Clostridia  Proteolytic clostridia degrade proteins released from dead organisms and ferment amino acids  Some strictly proteolytic, some both saccharolytic and proteolytic  Some ferment individual amino acids to yield a fatty acid– CoA derivative, then produce ATP via substrate-level phosphorylation with ammonia (NH3) and CO2  Stickland reaction: ferment amino acid pairs with one amino acid as electron donor and the other acceptor  Products are NH3 and CO2, and a carboxylic acid with one fewer carbon than the oxidized amino acid  Purines and pyrimidines from nucleic acid degradation lead to many of the same fermentation products and ATP through fatty acid–CoA derivatives 14 Use of energy-converting hydrogenases  Example: hyperthermophilic archaea Pyrococcus furiosus  Uses modified glycolysis, forming 3phosphoglycerate instead of 1,3bisphosphoglyceric acid  Forms ferrodoxinred and ATP from conversion of pyruvate to acetate  Uses energy-converting hydrogenases to achieve redox balance  Translocates Na+ across membrane, sodium motive force synthesizes ATP  Generates 4 ATP from substrate-level and oxidative phosphorylation, better than lactic acid bacteria 15 Primary vs secondary fermentations  Primary fermentations are carried out by organisms that break down and ferment carbohydrate, protein, fat polymers and monomers to reduced products, H2, CO2  Secondary fermentations use fermentation products as substrates for additional fermentation reactions  Products are mainly volatile fatty acids, H2, CO2  Propionibacterium, an important agent in the ripening of Emmental (Swiss) cheese, probably uses lactic acid, a fermentation product of lactic acid bacteria, as a major substrate to produce propionic acid (nutty taste of the cheese) and CO2 (holes in the cheese) 16 Fermentations that lack substrate-level phosprylation  Fermentations of certain compounds do not yield sufficient energy to synthesize ATP by substrate-level phosphorylation but support anaerobic growth  In these cases, catabolism is linked to ion pumps that establish a membrane gradient (e.g., succinate fermentation by Propionigenium modestum, oxalate fermentation by Oxalobacter formigenes) 17 Succinate fermentation: lacking substrate-level phosphorylation  Propionigenium modestum: requires NaCl for growth and succinate catabolism under strict anoxic conditions  An unusual decarboxylase, sodiumextruding decarboxylase, is involved in the fermentation  Succinate is converted to propionate through the decarboxylation of the intermediate (S)-methylmalonyl-CoA  Energy released by the decarboxylation reaction is used to translocate two Na+ across the membrane  Energy is then conserved by using a Na+ -linked ATPase 18 Syntrophy  Syntrophy: Two different microbes cooperate to perform a metabolic reaction neither can do alone  Most syntrophic reactions are secondary fermentations  Major compounds are fatty acids and alcohols  Interspecies Electron Transfer is the core of syntrophic reactions  One species serves as the electron acceptor for another species that is the electron donor  Electron transfer can be direct: through contact between cells; or  Mediated: through diffusion of metabolic products; e.g., H2 19 Interspecies H2 transfer  The ethanol fermentation carried out by the syntroph (Pelotomaculum) has a positive free energy; thus it cannot grow in pure culture  The H2 produced can be used as an electron donor by a methanogen to produce methane  The sum of the two reactions is exergonic; hence when cultured together, both organisms grow Overview of syntrophic transfer of H2 Reactions 20 Oxygenases in aliphatic hydrocarbon  Oxygenases catalyze the incorporation of O2 into organic (and some inorganic) compounds  Dioxygenases: incorporate both oxygen atoms  Monooxygenases: incorporate one oxygen atom with the second reduced to H2O  Usually dependent on NADH or NADPH  Oxygen atom is incorporated initially at a terminal carbon  End-product is a fatty acid of the same carbon length as the original hydrocarbon  Fatty acid is oxidized by beta-oxidation: removing two carbons at a time, forming NADH for electron transport, acetyl Co-A, and a fatty acid two carbons shorter  Acetyl-CoA is oxidized through the citric acid cycle or to make new cellular material 21 Multiple oxygenases in aromatics catabolism  Typically starts with formation of catechol or structurally related compound formation via oxygenases  Benzene to catechol using a hydroxylating monooxgenase  Toluene to methylcatechol by a hydroxylating dioxygenase  Catechol and related aromatics are cleaved by ring-cleavage dioxygenases  Degraded into compounds that can enter the citric acid cycle 22