BENG0004 2021 Lecture 18 Fermentation PDF

BENG0004 Biochemistry and Molecular Biology Emily Kostas Lecture 18 Fermentation  Fermentation Definition of fermentation: - a process where the terminal acceptor of reducing equivalents or terminal electron acceptor is...

BENG0004 Biochemistry and Molecular Biology Emily Kostas Lecture 18 Fermentation  Fermentation Definition of fermentation: - a process where the terminal acceptor of reducing equivalents or terminal electron acceptor is not O2 but an organic compound. The EMP Glycolytic pathway in a mammalian cell The cell has a limited amount of co-factors NAD and NADH In E. coli the total nicotinamide co-factor conc. is 1 mM, made up of about 0.8 mM NAD+, 0.02 mM NADH, 0.05 mM NADP+ and 0.15 mM NADPH This pair of co-factors are the major way cells have of moving reducing equivalents around [remember - reducing equivalents are the way energy is removed from the compounds, such as sugars, that we consume] The NADH must be recycled back to NAD reduction NADH NAD oxidation 1 mM total Nicotinamide cofactors in E. coli For energy generation and transfer of energy and catabolic pathways: 0.8 mM NAD+:0.02 mM NADH = 40:1 in the ox:red direction For anabolic (biosynthetic) reactions: 0.05 mM NADP+ : 0.15 mM NADPH = 1:3 in the ox:red direction So the cofactors for energy generation and catabolic pathways are poised to rapidly remove a proton and an electron from incoming substrates such as glucose.  Reoxidation of NADH during fermentation when no oxygen is present Glucose Glyceraldehyde-3-phosphate NAD+ NAD+ NADH + H+ NADH + H+ 1,3-bisphosphoglycerate NADH + H+ Pyruvate NAD+ NADH + H+ Lactate X NAD+ Y NADH from glycolysis is reoxidised by being used to reduce pyruvate or a pyruvate derivative (X). Either lactate or reduced product (Y) is the result. Human cells Glucose Cytoplasm e.g. muscle Sufficient O2 2x 2x ADP NAD+ ATP NADH + H+ Pyruvate Malate-Aspartate shuttle NADH + H+ NAD+ O2 TCA cycle Mitochondrion Electron transport H2O chain Human cells Glucose Cytoplasm e.g. muscle Insufficient O2 2x 2x ADP NAD+ ATP NADH + H+ LDH Pyruvate Lactate Mitochondrion LDH = lactate dehydrogenase  Alcoholic fermentation Saccharomyces cerevisiae (yeast) uses Embden Meyerhof Parnas pathway Zymomonas mobilis (bacterium) uses Entner Doudoroff pathway Glycolysis Pyruvate decarboxylase (not present in muscle) Glucose Pyruvate Acetaldehyde + CO2 Alcohol dehydrogenase Acetaldehyde Ethanol NADH NAD  Fermentation end products from different organisms Pyruvate is the key metabolite that other compounds are derived from Humans when no O2 Lactic acid bacteria E. coli is a mixed acid fermenter and will produce acetate, formate, ethanol and H2  Timeline history of the Earth First land plant Apes!Humans End of heavy meteorite 475 M yr 1 M yr Bombardment 3.9 G yr Multicellular Earliest organisms life 3.8 G yr First Eukaryotic 530 M yr Earth formed cell 2.1 G yr 4.6 Billion yr Oxygenic 65 M yr The Photosynthesis 3 G yr present Anaerobic atmosphere Oxygen atmosphere G yr = Billion yr all times are before present M yr = Million yr Currently many habitats exist where oxygen is limiting or not present: Our large intestine (and all other animals. About 2 kg bacteria in us) Gums Mud (bottom of ocean, lakes) Soil (after the first few cm) Silage Fermenting foods and beverages For organisms to survive and degrade sugars such as glucose, strategies of continued cycling of the NAD/NADH couple have evolved The organisms which can do this are bacteria and yeasts Glycolysis by Embden Meyerhof Parnas pathway - If O2 is present (aerobic glycolysis) and all NADH can feed into electron transport chain and thus O2 + H2O, then: Net ATP production: 1 Mole Glucose = 36 Mole ATP In anaerobic glycolysis: 1 Mole glucose = 2 Mole ATP (Glucose to Lactate) Other glycolytic pathways - Entner-Doudoroff pathway of glucose catabolism 1 Mole glucose = 1 Mole of ATP Phosphoketolase pathway of glucose catabolism 1 Mole glucose = 1Mole ATP Entner-Doudoroff pathway ATP Glucose ADP Glucose-6-phospate NADP NADPH 6-phosphogluconate H2O 2-keto-3 deoxy-6-phosphogluconate pyruvate glyceraldehyde-3-phosphate NAD NADH ADP Net result: ATP One ATP One NADPH One NADH ADP ATP pyruvate  Glycerol Widely used in industry and foods. During both world wars glycerol was produced by microbial processes. Yeast fermentation in presence of NaHSO3 (bisulphite) The bisulphite couples with the acetaldehyde making it unavailable for alcohol dehydrogenase The NADH which builds up is used by glycerol phosphate dehydrogenase to reduce dihydroxy acetone phosphate and produce glycerol phosphate The glycerol phosphate is rapidly de-phosphorylated by intracellular phosphatases giving glycerol Making glycerol by fermentation with Yeast and bisulphite (NaHSO3) Glucose Glyceraldehyde- Dihydroxyacetone- 3-phosphate phosphate NAD+ NADH 1,3 biphospho- glycerate The standard low O2 fermentation Acetaldehyde pathway to ethanol Ethanol Making glycerol by fermentation with Yeast and bisulphite (NaHSO3) Glucose Glyceraldehyde- Dihydroxyacetone- 3-phosphate phosphate NAD+ NADH 1,3 biphospho- Glycerol- glycerate phosphate A non-specific phosphatase OH NaHSO3 I -SO2Na X Acetaldehyde Bisulphite addition complex of acetaldehyde Glycerol Ethanol Fermented foods produced from fruits, vegetables and beans  Lactic acid An odorless, colorless liquid with a pleasant acid flavour. Used in many foods and soft drinks as a flavoring, a preservative and acidity regulator. As calcium lactate it is a convenient form of getting calcium into the body. The natural compound is L-lactate. For food use it has to be made by fermentation. Lactic acid bacteria are used. They are very widespread in the environment and have been harnessed by humans for thousands of years.  Uses of Lactic Acid Bacteria Fermented milk products (yogurts, cheeses, sauerkraut etc) 0.1% of the weight of cheese can be bacteria Silage - very widespread fermentation, carried out on large scale Many foods from around the world e.g sorghum, maize to produce ogi, cassava to produce gari and fufu, soybeans to produce tempeh, durian to produce tempoyak, Kimchi. Sourdough bread Silage - lactic acid fermentation on farms. Preserving grass for the winter months. Green plants are harvested and put into silos or black bags. A mixed population of bacteria present on green leafy material ferments carbohydrate rapidly and the pH falls to pH 4-5. The available oxygen is also rapidly consumed making the silage anaerobic. The temperature rises to about 30-500C The mixed population of bacteria present initially is gradually replaced by Lactobacilli species. The low pH effectively inhibits all toxin producing bacteria. [Lactate, acetate, proprionate - short chain fatty acids, benzoate all act as uncouplers and kill other bacteria]  Lactic Acid Bacteria Divided into two groups: Homofermentative (homolactic) Heterofermentative (heterolactic) Homofermentative e.g. Lactobacillus plantarum, L. casei, L. delbrueckii produce lactic acid only. They use the Embden-Meyerhof pathway of glycolysis from glucose to pyruvate. There is no pyruvate decarboxylase so no loss of CO2 NADH NAD+ Using L. delbrueckii at 500C in 13,000 litre fermenter - yield is 85-90% of available hexose (glucose). Over 20,000 tonnes of L+-lactic acid produced annually. a) b) a) Homolactic pathway b) Heterolactic pathway  Leuconostoc species Gram positive bacteria found in silage, on plants, in milk. Some species are used in wine production and in the fermentation of vegetables such as cabbage to produce sauerkraut and the fermentation of cucumbers to make pickles. They are also used in the manufacture of buttermilk and cheese. L. mesenteroides synthesises dextrans (extracellular polymer) from sucrose and is important in the industrial production of dextran. They use the phosphoketolase pathway to convert glucose to lactate, ethanol and acetate in varying amounts. The phosphoketolase pathway- a heterolactic fermentation pathway. e.g. in Leuconostoc species the Ribose pathway converts glucose to lactate, ethanol and CO2 Net 1 ATP Ribose-5-phosphate Acetate Sauerkraut Sauerkraut production employs a lactic acid fermentation. The basic process involves fermentation of shredded cabbage in the presence of 2.25- 2.5% by weight of salt to inhibit spoilage organisms. Rumen biochemistry - fermentation of cellulosic plant material Volume of rumen = 60 litres Contains 5x1010 bacteria/ml 1x106 protozoa Some yeasts and fungi Fibrobacter sp.  Bloodstream to cells Mouth 3 x 1016 bacteria per rumen; 6 x 1010 protozoa per rumen Starch Cellulose Pectin Hemicellulose Xylose Galacturonic acid