Fermentation in the Bacterial Psychology | HSCI 4607, Study notes of Health sciences

Download Fermentation in the Bacterial Psychology | HSCI 4607 and more Study notes Health sciences in PDF only on Docsity! Bacterial Physiology HSCI 4607/5607 Ch: 14 Fermentation Introduction: Many prokaryotes can grow indefinitely in the complete absence of oxygen unlike the eukaryotes. These organisms re-oxidize reduced coenzymes like NADH either by anaerobic respiration using nitrate, sulfate or fumerate as a electron acceptor or by fermentation. Fermentation can be defined as a pathway in which the reduced coenzymes are oxidized by cellular metabolites produced in some pathway. This redox reaction takes place in the cytosol rather than in the membrane and ATP is produced by substrate level phosphorylation. Fermentations are named after the major end products they generate. For example ethanol fermentation carried out by yeast and lactic acid fermentation by muscle and red blood cells. The carbohydrate fermentations can be divided into six different classes: 1. Lactic acid; 2. Ethanol; 3. Butyric; 4. Mixed acid; 5. Propionic; and 6. Homoacetic fermentations. A Substrate level phosphorylation 1. 1,3 BPGA + ADP ————> 3-PGA + ATP. 2. PEP + ADP ——————> pynvate + ATP 3. acetyl-P + ADP —-———_> acetate + ATP B Fumarate respiration IN OUT H*+NADH 2 Ht + fumarate succinate: NADH is e- donor Periplasmic e- donor (AH,) © Proton-coupled (or Na*-coupled) © D —_Na*-dependent decarboxylation end-product efflux of an organic acid IN ‘i OUT lactate > nt — > E Electrogenic oxalate: IN format exchange our “O,C;C0} << "0,0-CO} = aj Oxalobacter HCOoO-—_ <\ >  Electron sinks: The reduced coenzymes which are the byproducts of the oxidative metabolic pathways are reoxidized in respiratory organisms by either oxygen or nitrate. In case of fermentation, the exogenous supply of electron acceptor is not there, therefore the fermentative metabolic pathways have to provide electron acceptors. During fermentation these electron acceptors are reduced and often excreted out in the medium in large quantity, e.g. ethanol, organic acids, and solvents. Since these metabolites help to dispose of electrons from the reduced NADH, they are called ‘electron sinks’. Many times hydrogen gas is produced when protons are used as electron acceptors. Anaerobic Food Chain: Fermentative bacteria under anaerobic conditions convert amino acids, purines, pyrimidines and carbohydrates to organic acids, alcohols, H2, and CO2. These end products are converted to the gaseous end products like methane and CO2 which are in turn used by several bacteria and plants through out the biosphere. These steps are the part of carbon cycle and the conversions are carried out by the action of several groups of bacteria which utilize various products as carbon source and complete the conversion to continue the cycle. This is known as anaerobic food chain. Generally this takes place in the anaerobic environments like muds, bottom of the lakes, and sewage treatment plants. Propionate Fermentation via Succinate-propionate Pathway: Many bacteria produce propionic acid via succinic acid as an intermediate instead of acrylate. This pathway yields more ATP per mole of propionate formed than acrylate pathway. The organisms that use this pathway ferment lactate and hexoses to form propionate, acetate, and CO2. The example is Propionibacterium which is a gram positive, non-sporulating, nonmotile, pleomorphic rods, a part of the normal flora of rumen in herbivores, human skin and in dairy products. Propionibacterium is used in the production of swiss cheese and it imparts sharp flavor which is due to propionate and holes are due to the production of CO2. The pathway generates more ATPs and protons which are used to generate p. The Succinate -Propionate Path way: 6[H] 0 arn) [CO 9 > a 3 CH;-CHOH—CHOH fe 3 cH,-C—cooa 4 > CH,-C~ SCoA —i- CH,-C-O~(P) lactate pyruvate acetyl-CoA acetyl-P 2 CH;-CH,-COOH “ 0 ‘ar fonat tl A propionate f 2102] 2 HOOC—CH ,-C—COOH a aim oxaloacetate CH;COOH Il 2 CHy-CH,-C~SCoA propionyl-CoA 2. HOOC—CH,-CHOH—COOH 7 malate 2,0 CH ? 2 HOOC—CH=CH—COOH 2 HOOC~CH-C~SCoA femigeate methyl- ADP +P, malonyl-CoA 4{H) > ee h 2(CoASH]) {I 2 HOOC-CH,-CH,-C~SCoA <—>— 2 HOOC—CH,-CH,—COOH succinyl-CoA succinate Fig. 14.4 Propionate fermentation by the succinate-propionate pathway. Enzymes: 1, lactate dehy- drogenase (a flavoprotein); 2, pyruvate dehydrogenase (an NAD* enzyme); 3, phosphotransacetylase; 4, acetate kinase; 5, methylmalonyl-CoA-pyruvate transcarboxylase; 6, malate dehydrogenase; 7, fumarase; 8, fumarate reductase; 9, CoA transferase; 10, methylmalony|—-CoA racemase.  PEP Carboxyphosphorylase Reaction: Propionibacteria can produce succinate as well as propionate when grown on carbon source such as glucose via glycolytic pathway. In this case the C3 intermediate, Phosphoenolpyruvate is carboxylated to oxaloacetate by an enzyme PEP carboxytransphosphorylase : PEP + CO2 +Pi > Oxaloacetate +PPi Oxaloacetate is than converted by the reactions 6, 7, 8 of succinate pathway to succinate and PPi acts as a phosphate donor to phosphorylate Fructose-6-P to Fructose 1,6 biP or serine to phosphoserine. There are several physiological advantages of this pathway: 1. It generates fumerate which acts as a electron sink to oxidize NADH; 2. The fumerate reductase is a coupling site to generate p.; and 3. The succinate can be converted to succinyl CoA, which is required for the biosynthesis of tetrapyrroles, lysine, diaminopimelic acid, and methionine. Lactate Fermentation: The lactic acid bacteria are the heterogeneous group of aerotolerant organisms that ferment glucose to lactic acid even under aerobic conditions. These organisms include the genera, Lactobacillus, Sporolactobacillus, Strptococcus, Leuconostoc, Pediococcus, and Bifidobacterium. These organisms are the normal habitats of skin of animals, gastrointestinal tract, mouth and throat and also dairy products. Some of these organisms are medically as well as commercially very important since some are pathogenic, e.g genus Streptococcus while many are used for the production of fermented dairy (yogurt and cheese) or other products like pickles, and saurkraut. They are aerotolerant but derive their energy by fermenting glucose and via substrate level phosphorylation. Under some conditions they also transport lactate and create p. There are two major pathways through which bacteria produce lactate: 1. Homofermentation which uses Embden-Meyerhof-Parnas pathway (Glycolysis); 2. Hetero-fermentative which uses Pentose phosphate pathway. There is also a third pathway which is used only by Bifidobacterium bifidum known as Bifidum pathway. Homofermentative Lactate Fermentation: The homofermentative pathways produce primarily lactic acid through the glycolytic pathway. Glucose is converted to two molecules of pyruvate with the net yield of two ATPs. The NADH molecules which are produced are used to reduce pyruvate to lactate. The overall reaction is: Glucose + 2ADP +2Pi > 2 Lactate + 2 ATP Heterofermentative Lactate Fermentation: The heterofermentative pathway uses decarboxylation and isomerization reactions of pentose phosphate pathway. Xylulose-5-P is cleaved with the help of inorganic phosphate by enzyme phosphoketolase to phosphoglyceraldehyde and acetyle-P using thiamine pyrophosphate as cofactor. The phosphoglyceraldehyde is oxidized to pyruvate (glycolysis) yielding ATP and NADH. Pyruvate is then reduced to lactate by NADH. The acetyl phosphate is reduced to ethanol using two NADHs. The overall reaction is: Glucose + ADP + Pi > Ethanol + Lactate + CO2 + ATP 2(H] 2pHy (C glucose —ATP » Gep i 6-P-gluconate 4+ RuMP ‘cH,OH ro HoH H“c—0H z tH0® xyl-5-P Ns H—c=0 ‘cits ‘om {o-® ‘cH,O®) O acetyl-P Pj PGALD CoASH 2{H] <- Fj ie I ' c-o~@) § SCoA CHOH ° CHO ®) 20H] apr, 1,3 BPGA ATP: CH, H-C=0 COOH acetaldehyde CHOH anh CHOP) 3-PGA qs HO p ATP gooH COOH bo <i ro ¢=0 E> cio ¢H,0H cu dy cH. [iactate| PGA ° PEP pyruvate : ie Fig. 14.6 Heterofermentative lactate fermentation. Enzymes: 1, hexokinase; 2, glucose-6-phosphate dehydrogenase; 3, 6-phosphogluconate dehydrogenase; 4, ribulose-5-phophate epimerase; 5, phos- phoketolase; 6, phosphotransacetylase; 7, acetaldehyde dehydrogenase; 8, alcohol dehydrogenase; 9, PGALD dehydrogenase; 10, PGA kinase; 11, phosphoglycerate mutase; 12, enolase; 13, pyruvate kinase; 14, lactate dehydrogenase.  Mixed Acid and Butanediol Fermentation: Facultative anaerobic enteric bacteria undergo several physiological changes to adapt anaerobic conditions. These include: 1. Terminal reductases replace the oxidases in the ETC. 2. The citric acid cycle is modified to become a reductive pathway. -Ketoglutarate dehydrogenase and succinate dehydrogenase activity disappear or present at low level. The activity is replaced by fumerate reductase. 3. Pyruvate-formate lyase is substituted for pyruvate dehydrogenase. Thus the cells oxidize pyruvate to acetyl-1-CoA and formate, rather than to acetyl-CoA, CO2, and NADH. 4. They carry out mixed acid or butanediol fermentation. Both butanediol and mixed acid fermentations are same in that they produce mixture of organic acids, CO2, H2, and ethanol. In butandiol fermentation, large amounts of 2,3-butandiol, acetoin, more CO2, and ethanol, and less acid is produced. Mixed acid producers belong to the genera Escherichia, Salmonella, and Shigella. All of them are intestinal pathogens. Butandiol fermenters are Serratia, Erwinia, and Enterobacter. Mixed Acid Fermentation: The products of mixed acid fermentation are succinate, lactate, acetate, ethanol, formate, CO2, and hydrogen gas. Each of these products are made from one phosphoenol- pyruvate and CO2 or one pyruvate. For example, the formation of succinate is due to a carboxylation of phosphoenolpyruvate to oxaloacetate followed by two reductions to form succinate. All other products are formed from pyruvate. Lactate is formed by reduction of pyruvate. Pyruvate is also decarboxylated to acetyl-CoA and formate by pyruvate-formate lyase. The acetyl-CoA can be converted to ethanol by reduction or may be converted to acetate via acetyl-P. The format can be oxidized to CO2 and H2 by format- hydrogen lyase. 10 PEP P; 2|-ADP ATP OAA pyruvate i} 1 3/CoASH malate acetyl-CoA + [formate ] [formate | CO, 12 xe 5 I a i cm Lik [Ha] fumarate 13/2[H] succinate Fig. 14.7 Mixed acid fermentation. Enzymes: 1, glycolytic enzymes; 2, pyruvate kinase; 3, pyruvate— formate lyase; 4, lactate dehydrogenase; 5, formate-hydrogen lyase; 6, acetaldehyde dehydrogenase; 7, alcohol dehydrogenase; 8, phosphotransacetylase; 9, acetate kinase; 10, PEP carboxylase; 11, malate dehydrogenase; 12, fumarase; 13, fumarate reductase. Note the ATP yields: per succinate, approximately 1; per ethanol, 1; per acetate, 2; per formate, 1; per CO2 and Ha, 1; per lactate, 1. Energy equivalent to approximately 1 ATP is conserved per succinate formed because the fumarate reductase reaction takes place in the cell membrane and generates a Ap. Note also the reducing equivalents used in the production of the end products: per succinate, 4; per ethanol, 4; per acetate, 0; per lactate, 2; per formate, 0, The number of reducing equivalents used must equal the number produced during glycolysis. Therefore, only certain ratios of end products are compatible with a balanced fermentation.  Butyrate Fermentation: Some bacteria belonging to genus Clostridium produce butyric acid via fermentation. These organisms are anaerobic spore-forming and commonly found in muds, sewage, feces or other anaerobic environment. The butyric acid clostridia ferment carbohydrates to butyric acid. The other byproducts include H2, CO2, and small amount of acetate. Glucose is first oxidized to two moles of pyruvate through glycolysis with two molecules of NADH and two ATPs. Pyruvate is then decarboxylated to acetyl-CoA, CO2, and H2 using pyruvate-ferredoxin oxidoreductase and hydrogenase. The acetyl-CoA is condensed to form acetoacetyl-CoA, which is reduced to -hydroxybutyryl-CoA using two NADHs. The CoASH is then displaced by inorganic phosphate to produce butyryl phosphate, which donates phosphoryl group to ADP to form ATP and Butyrate. There are three substrate level phophorylation including two during glycolysis. lase);4, -hydroxybutyryl CoA dehydrogenase; 5, crotonase; 6, butyryl CoA dehydrogenase; 7, Phosphotrans butyrylase; 8, Butyrate kinase; 9, Hydrogenase Butyrate and Butanol-Acetone Fermentation by C.acetobutylicum: Some butyrate making clostridia switch to Butanol-acetone fermentation when butyrate level reaches to certain level and pH of the medium drops. The organisms actually take up butyrate and convert it to butanol and acetone. This is perhaps a rescue mechanism since excess of undissociated butyrate at low pH being lipophilic can enter the cell and interfere with growth process (uncoupler). Butanol-Acetone production was once the second largest industrial fermentation process, second only to ethanol. Now they are synthesized chemically. For example in C.acetobutylicum during exponential phase it produces butyrate, acetate, CO2 and H2. This phase is termed as acidogenic phase. When organism enters into stationary phase it takes up butyrate and with the fermentation of carbohydrates, it converts it to butanol, acetone and ethanol. This phase is termed as ‘Solvent phase’. The molar ratio of butanol-acetone varies in different strains, but in C.acetobutylicum this ratio is 2:1 with the small amount of isopropanol.