Biology 120 lectures for 2nd exam 2012 2012

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  • 1. MICROBIAL GROWTH AY 2012-2013Monday, September 3, 2012
  • 2. DEFINITION OF MICROBIAL GROWTH • NUMBER OF CELLS • NOT CELL SIZE • e.g. Growing microbes = increase in numbers, accumulating coloniesMonday, September 3, 2012
  • 3. DEFINITION OF MICROBIAL GROWTH • Note: for coenocytic organisms (multinucleate): growth = increased cell sizeMonday, September 3, 2012
  • 4. FOR YOU TO GROW....Monday, September 3, 2012
  • 5. HOW ABOUT THEM?Monday, September 3, 2012
  • 6. HOW ABOUT THEM?Monday, September 3, 2012
  • 7. HOW ABOUT THEM?Monday, September 3, 2012
  • 8. RECALL MICROBIAL NUTRITION CARBON SOURCES Autotrophs CO2 sole or principal biosynthetic carbon source Heterotrophs Reduced, preformed, organic molecules from other organisms ENERGY SOURCES Phototrophs Light Chemotrophs Oxidation of organic or inorganic compounds HYDROGEN AND ELECTRON SOURCES Lithotrophs Reduced inorganic molecules Organotrophs Organic moleculesMonday, September 3, 2012
  • 9. RECALL MICROBIAL NUTRITION MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, REPRESENTATIVE HYDROGEN/ELECTRONS AND MICROORGANISMS CARBON PHOTOLITHOTROPHIC Light energy Algae AUTOTROPHY Inorganic hydrogen/electron Purple and green sulfur donor bacteria CO2 carbon source Blue-green algae (cyanobacteria) PHOTOORGANOTROPHIC Light energy Purple non-sulfur bacteria HETEROTROPHY Organic hydrogen/electron Green non-sulfur bacteria donor Organic carbon source (CO2 may also be used)Monday, September 3, 2012
  • 10. RECALL MICROBIAL NUTRITION MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, REPRESENTATIVE HYDROGEN/ELECTRONS AND MICROORGANISMS CARBON CHEMOLITHOTROPHIC Chemical energy source Sulfur-oxidizing bacteria AUTOTROPHY (inorganic) Hydrogen bacteria Inorganic hydrogen/electron Nitrifying bacteria donor Iron bacteria CO2 carbon source CHEMOORGANOTROPHIC Chemical energy source Protozoa HETEROTROPHY (organic) Fungi Organic hydrogen/electron Most non-photosynthetic donor bacteria Organic carbon sourceMonday, September 3, 2012
  • 11. REQUIREMENTS FOR MICROBIAL GROWTH •PHYSICAL •CHEMICAL REQUIREMENTS REQUIREMENTS • TEMPERATURE • CARBON • • pH NITROGEN, SULFUR & PHOSPHORUS • OSMOTIC • TRACE ELEMENTS PRESSURE • OXYGEN • ORGANIC GROWTH FACTORSMonday, September 3, 2012
  • 12. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATURE • “Most microorganisms grow well at temperatures favored by humans” • 3 primary groups (on the basis of temperature preference) • psychrophiles (cold-loving) • mesophiles (moderate-temperature-loving) • thermophiles (heat-loving)Monday, September 3, 2012
  • 13. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATURE MINIMUM, OPTIMUM, MAXIMUMMonday, September 3, 2012
  • 14. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATURE • Psychrotrophs: grow between 0°C and 20-30°C; cause food spoilage • Hyperthermophiles : extreme temperatures (members of the archaea)Monday, September 3, 2012
  • 15. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATUREMonday, September 3, 2012
  • 16. REQUIREMENTS FOR MICROBIAL GROWTH: pH • RECALL: pH acidity or alkalinity of a solution • acidophiles • neutrophiles • alkaliphilesMonday, September 3, 2012
  • 17. REQUIREMENTS FOR MICROBIAL GROWTH: OSMOTIC PRESSURE • Reactions of microorganism in solution based on solute concentration: hypertonic, isotonic, hypotonic • e.g. based on osmotic pressure requirement: Halophiles (obligate/extreme or facultative) • Water activity (aw): water that is available for metabolic processes; i.e. water in food which is not bound to food molecules can support the growth of bacteria, yeasts and molds (fungi) or unbound and available waterMonday, September 3, 2012
  • 18. REQUIREMENTS FOR MICROBIAL GROWTH: OSMOTIC PRESSUREMonday, September 3, 2012
  • 19. REQUIREMENTS FOR MICROBIAL GROWTH: CARBON • one of the most important requirements for microbial groth • structural backbone of living matter • e.g. Chemoautotrophs (carbon dioxide) and Chemoheterotrophs (organic materials)Monday, September 3, 2012
  • 20. REQUIREMENTS FOR MICROBIAL GROWTH: NITROGEN • ACCESS: amino acids and proteins • Most bacteria decompose proteins • Some bacteria use NH4+ or NO3– • A few bacteria use N 2 in nitrogen fixationMonday, September 3, 2012
  • 21. REQUIREMENTS FOR MICROBIAL GROWTH: SULFUR • ACCESS: amino acids, thiamine and biotin • Most bacteria decompose proteins • Some bacteria use SO42– or H2SMonday, September 3, 2012
  • 22. REQUIREMENTS FOR MICROBIAL GROWTH: NITROGEN, SULFUR AND PHOSPHORUS • ACCESS: In DNA, RNA, ATP and membranes • PO is a 4 3– source of phosphorusMonday, September 3, 2012
  • 23. REQUIREMENTS FOR MICROBIAL GROWTH: TRACE ELEMENTS • iron, copper, molybdenum, zinc • essential for the function of co- factorsMonday, September 3, 2012
  • 24. REQUIREMENTS FOR MICROBIAL GROWTH: TRACE ELEMENTS • BIOTIN • PYRIDOXINE or VIT B6 • Carboxylation (Leuconostoc) • Transamination (Lactobacillus) • CYANOCOBALAMIN or VIT B12 • NIACIN • Molecular rearrangements (Euglena) • Precursor of NAD and NADP (Brucella) • FOLIC ACID • RIBOFLAVIN or VIT B2 • One-carbon metabolism (Enterococcus) • Precursor of FAD and FMN (Caulobacter) • PANTOTHENIC ACID • THIAMINE or VIT B1 • Fatty acid metabolism (Proteus) • Aldehyde group transfer (BacillusMonday, September 3, 2012
  • 25. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • “microbes that use molecular oxygen produce more energy from nutrients than microbes that do not use oxygen”Monday, September 3, 2012
  • 26. Monday, September 3, 2012
  • 27. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • aerobic bacteria • anaerobic bacteria • microaerophilic bacteriaMonday, September 3, 2012
  • 28. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • Microbes can be harmed by toxic forms of oxygen • singlet oxygen ( 2 1O -): normal molecular oxygen that has been boosted into a higher- energy state; extremely reactive • hydroxyl radical (OH•): most reactive intermediate form of oxygen formed in cellular cytoplasm by ionizing radiationMonday, September 3, 2012
  • 29. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • Microbes can be harmed by toxic forms of oxygen • peroxide anion (O22-): toxic; active ingredient in hydrogen peroxide and benzoyl peroxide • SOLUTION: catalase and peroxidaseMonday, September 3, 2012
  • 30. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • Microbes can be harmed by toxic forms of oxygen • superoxide free radicals (O2-): toxicity is caused by their great instability; they steal an electron from a neighboring molecule, which in turn becomes a free radical, and the cycle continues • SOLUTION: production of superoxide dismutase (SOD): aerobic, FA and aerotolerant anaerobes • convert superoxide free radicals to molecular oxygen and hydrogen peroxideMonday, September 3, 2012
  • 31. REQUIREMENTS FOR MICROBIAL GROWTH: ORGANIC GROWTH FACTORS • VITAMINS: Unlike humans, most bacteria can synthesize all their own vitamins and are not dependent on outside sources • Some bacteria lack the enzymes needed for the synthesis of certain vitamins, amino acids, purines and pyrimidinesMonday, September 3, 2012
  • 32. REVISED SCHEDULE DATE ACTIVITY August 28 (2 hour) Microbial Growth and Metabolism/Physiology August 28-September 4 Metabolism and Physiology and Microbial Control (4 hours) September 11 (2 hours) Journal Reporting Group 2 (10 pairs) September 18 (2 hours) EXAMINATION 2 September 25 Microbial Genetics October 2 (2 hours) Microbial Interactions October 9 (2 hours) EXAMINATION 3Monday, September 3, 2012
  • 33. CULTURE MEDIAMonday, September 3, 2012
  • 34. CULTURE MEDIA • nutrient material prepared for the growth of microorganisms in a laboratory • IMPORTANT TERMS: • inoculum: microbes introduced into a culture medium • culture: microbes that grow and multiply in a culture medium • sterile medium: a pre-requisite = no living microorganismsMonday, September 3, 2012
  • 35. AGAR • solidifying agent • only a few microbes can degrade it • liquifies at 1000C and solidifies below 400C • pouring temperature: 500C (prevents injury to microbes) • used for the preparation of slants, stabs/deeps, platesMonday, September 3, 2012
  • 36. TYPES OF CULTURE MEDIA: Chemically-defined Media • exact chemical composition is known • mostly for autotrophic bacteria, fastidious bacteria • Contents: organic growth factors (carbon and energy)Monday, September 3, 2012
  • 37. TYPES OF CULTURE MEDIA: Complex Media • made up of nutrients including extracts from yeasts, meat or plants, or digests of proteins • exact chemical composition varies from batch to batch • mostly for heterotrophic bacteria and fungiMonday, September 3, 2012
  • 38. TYPES OF CULTURE MEDIA: Anaerobic Growth Media • “reducing media” • sodium thioglycollate: chemically combine with dissolved oxygen and deplete the oxygen in the culture medium • heated first before use to drive off absorbed oxygenMonday, September 3, 2012
  • 39. ANAEROBIC CULTURE TECHNIQUESMonday, September 3, 2012
  • 40. ANAEROBIC CULTURE TECHNIQUESMonday, September 3, 2012
  • 41. ANAEROBIC CULTURE TECHNIQUESMonday, September 3, 2012
  • 42. TYPES OF CULTURE MEDIA: Selective & Differential Media • Goal: to detect the presence of specific microorganisms associated with disease or poor sanitation • SELECTIVE: suppress growth of unwanted bacteria and encourage the growth of desired microbesMonday, September 3, 2012
  • 43. TYPES OF CULTURE MEDIA: Selective & Differential Media •Why it can select: • BSA: Bismuth Sulfite Indicator and Brilliant Green are complementary, inhibiting Gram- positive bacteria and coliforms, allowing Salmonella spp. to grow • SDA: pH 5.6 where fungi can outgrow bacteriaMonday, September 3, 2012
  • 44. TYPES OF CULTURE MEDIA: Selective & Differential Media • Goal: to detect the presence of specific microorganisms associated with disease or poor sanitation • DIFFERENTIAL: distinguish colonies of desired organisms when grown together with othersMonday, September 3, 2012
  • 45. TYPES OF CULTURE MEDIA: Differential MediaMonday, September 3, 2012
  • 46. TYPES OF CULTURE MEDIA: Differential MediaMonday, September 3, 2012
  • 47. TYPES OF CULTURE MEDIA: Enrichment Media • mostly for soil and fecal samples or when desired microbe is injured • may also be selective • e.g. MRS agar (deMann, Rogosa and Sharpe agar or Lactobacillus agar) • e.g. lactose brothMonday, September 3, 2012
  • 48. PURE CULTUREMonday, September 3, 2012
  • 49. PREPARING PURE CULTURE • Julius Richard Petri (1887) • Easy to use, stackable (saving space), requirement for plating methodsMonday, September 3, 2012
  • 50. OBTAINING PURE CULTURES: Streak PlatingMonday, September 3, 2012
  • 51. PURE VS MIXED CULTUREMonday, September 3, 2012
  • 52. CHARACTERIZING COLONIESMonday, September 3, 2012
  • 53. CULTURE PRESERVATIONMonday, September 3, 2012
  • 54. WAYS TO PRESERVE YOUR CULTURE •subculturing • mineral oil overlay • freezing as glycerol stocks • liquid nitrogen storage • lyophilizationMonday, September 3, 2012
  • 55. WAYS TO PRESERVE YOUR CULTURE • subculturing •mineral oil overlay • freezing as glycerol stocks • liquid nitrogen storage • lyophilizationMonday, September 3, 2012
  • 56. WAYS TO PRESERVE YOUR CULTURE • subculturing • mineral oil overlay •freezing as glycerol stocks • liquid nitrogen storage • lyophilizationMonday, September 3, 2012
  • 57. WAYS TO PRESERVE YOUR CULTURE • subculturing • mineral oil overlay • freezing as glycerol stocks •liquid nitrogen storage • lyophilizationMonday, September 3, 2012
  • 58. WAYS TO PRESERVE YOUR CULTURE • subculturing • mineral oil overlay • freezing as glycerol stocks • liquid nitrogen storage •lyophilizationMonday, September 3, 2012
  • 59. REVIVAL OF PRESERVED L- DRIED CULTURES http://www.jcm.riken.jpMonday, September 3, 2012
  • 60. GROWTH OF BACTERIAL CULTURESMonday, September 3, 2012
  • 61. BACTERIAL DIVISIONMonday, September 3, 2012
  • 62. OTHER FORMS OF DIVISION BY OTHER MICROBES Budding = Chains of conidiospores Rhodopseudomonas carried externally at the tips of the filaments = Actinomycetes Fragmentation of filaments = ActinomycetesMonday, September 3, 2012
  • 63. THE MATHEMATICS OF GROWTHMonday, September 3, 2012
  • 64. CELL DIVISION • Generation time: time required for a microbial population to double • g = mean generation time • g = t/nMonday, September 3, 2012
  • 65. GENERATION TIME • g = t/nMonday, September 3, 2012
  • 66. SAMPLE... • Given an initial • Solution: t = 2 density of 4 x 104 • n = [ log (1 x 10 ) – 6 • After 2 hours the log (4 x10 4)]/ cell density became 0.301; n = 4.65 1 x 10 6 • Generation time = • Compute for the (t/n); 2/4.65 or 0.43 generation time hours OR 25.8 minutesMonday, September 3, 2012
  • 67. GENERATION TIME MICROORGANISM TEMPERATURE (°C) GENERATION TIME (hours) Escherichia coli 40 0.35 Bacillus subtilis 40 0.43 Mycobacterium 37 12 tuberculosis Euglena gracilis 25 10.9 Giardia lamblia 37 18 Sacharomyces 30 2 cerevisiaeMonday, September 3, 2012
  • 68. THE GROWTH CURVEMonday, September 3, 2012
  • 69. OBTAINING A GROWTH CURVE • The Growth Curve can be obtained via a Batch Culture • Microorganisms are cultivated in a liquid medium and grown as a closed system • Incubated in a closed culture vessel with a single batch of medium and NO fresh medium provided during incubation • SCENARIO: Nutrient concentration decline and concentrations of waste increase during the incubation periodMonday, September 3, 2012
  • 70. 1. THE LAG PHASE • No immediate increase in cell mass or cell number • Cell is synthesizing new components • Cells retool, replicate their DNA, begin to increase in mass and finally divideMonday, September 3, 2012
  • 71. 1. THE LAG PHASE • The necessity of a lag phase: • Cells may be old and ATP, essential cofactors and ribosomes depleted • must be synthesized first before growth can begin • Medium maybe different from the one the microorganism was growing previously • new enzymes would be needed to use different nutrients • Microorganisms have been injured and require time to recoverMonday, September 3, 2012
  • 72. SHORT LAG PHASE • SHORT LAG PHASE (or even absent) • Young, vigorously growing exponential phase culture is transferred to fresh medium of same compositionMonday, September 3, 2012
  • 73. LONG LAG PHASE • LONG LAG PHASE • Inoculum is from an old culture • Inoculum is from a refrigerated source • Inoculation into a chemically-different mediumMonday, September 3, 2012
  • 74. 2. THE LOG/ EXPONENTIAL PHASE • Microorganisms are growing and dividing at the maximal rate possible given their genetic potential, nature of medium and conditions under which they are growing • Rate of growth is constant: doubling at regular intervals • The population is most uniform in terms of chemical and physiological properties • Why the curve is smooth: • Because each individual divides at a slightly different momentMonday, September 3, 2012
  • 75. 3. STATIONARY PHASE • Population growth ceases and the growth curve becomes horizontal (around 109 cells on the average) • Why enter the stationary phase: • Nutrient limitation (slow growth) • Oxygen limitation • Accumulation of toxic waste productsMonday, September 3, 2012
  • 76. 4. DEATH PHASE • Detrimental environmental changes like nutrient depletion and build up of toxic wastes lead to the decline in the number of viable cells • Usually logarithmic (constant every hour) • DEATH: no growth and reproduction upon transfer to new medium • NOTE: Death rate may decrease after the population has been drastically reduced due to resistant cellsMonday, September 3, 2012
  • 77. DIRECT MEASUREMENT • Plate counts • Filtration • Most Probable Number (MPN) • Direct Microscopic CountMonday, September 3, 2012
  • 78. PLATE COUNTSMonday, September 3, 2012
  • 79. RECALL: HOW TO COMPUTE CFUMonday, September 3, 2012
  • 80. FILTRATIONMonday, September 3, 2012
  • 81. MPNMonday, September 3, 2012
  • 82. DMCMonday, September 3, 2012
  • 83. INDIRECT MEASUREMENTS: ESTIMATING BACTERIAL NUMBERS • Turbidity: spectrophotometry estimates • Metabolic Activity • e.g. MBRT for Milk = Class 1. Excellent, not decolorized in 8 hours; Class 2. Good, decolorized in less than 8 hours but not less than 6 hours; Class 3. Fair, decolorized in less than 6 hours but not less than 2 hours; Class 4. Poor, decolorized in less than 2 hours • Dry Weight: for filamentous moldsMonday, September 3, 2012
  • 84. MICROBIAL METABOLISM & PHYSIOLOGYMonday, September 3, 2012
  • 85. MICROBIAL METABOLISM • IMPORTANT: • most of the biochemical processes of bacteria also occur in eukaryotes • BUT...the reactions that are unique to bacteria are fascinating because they allow microorganisms to do things we cannot do • e.g. cellulose metabolism, petroleum metabolism or just iron, just hydrogen gas or just ammoniaMonday, September 3, 2012
  • 86. RECALLING THE BASICSMonday, September 3, 2012
  • 87. DEFINITION• METABOLISM: The sum of the chemical reactions in an organism• CATABOLISM: The energy-releasing processes• ANABOLISM: The energy-using processesMonday, September 3, 2012
  • 88. THE ROLE OF ATP • facilitates the coupling of anabolic and catabolic reactions • In Catabolism: some energy is transferred to and trapped in ATP and the rest given off as heat • In Anabolism: ATP provides the energy for synthesis and the rest given off as heatMonday, September 3, 2012
  • 89. ENZYMESMonday, September 3, 2012
  • 90. ENZYMES & THE COLLISION THEORY • Collision Theory: explains how chemical reactions occur and how certain factors affect the rates of those reactions • BASIS: all atoms, ions and molecules are continuously moving and colliding with one another • THUS: the energy transferred by the particles in the collision can disrupt their electron structures enough so that chemical bonds are broken or new bonds are formedMonday, September 3, 2012
  • 91. FACTORS THAT DETERMINE WHETHER A COLLISION WILL CAUSE A CHEMICAL REACTIONMonday, September 3, 2012
  • 92. FACTORS THAT DETERMINE WHETHER A COLLISION WILL CAUSE A CHEMICAL REACTION • velocities of colliding particles: higher velocities; greater chances of collision that will cause a reactionMonday, September 3, 2012
  • 93. FACTORS THAT DETERMINE WHETHER A COLLISION WILL CAUSE A CHEMICAL REACTION • velocities of colliding particles: higher velocities; greater chances of collision that will cause a reaction • their energy: requires a specific level of energyMonday, September 3, 2012
  • 94. FACTORS THAT DETERMINE WHETHER A COLLISION WILL CAUSE A CHEMICAL REACTION • velocities of colliding particles: higher velocities; greater chances of collision that will cause a reaction • their energy: requires a specific level of energy • their specific chemical configurations: no reaction will take place unless the particles are properly oriented toward each otherMonday, September 3, 2012
  • 95. ACTIVATION ENERGY & REACTION RATES • ACTIVATION ENERGY: amount of energy needed to disrupt the stable electronic configuration of any specific molecule so that the electrons can be rearranged • REACTION RATES: frequency of collisions containing sufficient energy to bring about a reactionMonday, September 3, 2012
  • 96. ENZYMES & CHEMICAL REACTIONS • Enzymes speed up chemical reactions (biological catalysts)Monday, September 3, 2012
  • 97. SPECIFICITY & EFFICIENCY • Specificity of enzymes is made possible by their structure • generally large globular proteins • 3D shape with a specific surface configuration • Enzymes are extremely efficient • turnover number (substrate to product conversion) = between 1-10, 000 (max 500,000)Monday, September 3, 2012
  • 98. • names will usually end in -ase and grouped according to type of chemical reaction they catalyzeMonday, September 3, 2012
  • 99. COMPONENTS OF ENZYMES Coenzyme: assist the enzyme by accepting atoms removed from the substrate or by donating atoms required by the substrate Important Coenzymes: NAD+, NADP+ , FAD and Coenzyme AMonday, September 3, 2012
  • 100. MECHANISM OF ENZYME ACTIONMonday, September 3, 2012
  • 101. FACTORS INFLUENCING ENZYME ACTIVITYMonday, September 3, 2012
  • 102. FACTORS INFLUENCING ENZYME ACTIVITY • Temperature • pH • Substrate concentration • InhibitorsMonday, September 3, 2012
  • 103. FACTORS INFLUENCING ENZYME ACTIVITY • Temperature • pH • Substrate concentration • InhibitorsMonday, September 3, 2012
  • 104. FACTORS INFLUENCING ENZYME ACTIVITY • Temperature • pH • Substrate concentration • InhibitorsMonday, September 3, 2012
  • 105. FACTORS INFLUENCING ENZYME ACTIVITY • Temperature • pH • Substrate concentration • Inhibitors Sulfanilamide as inhibitor of PABA during folate synthesis in bacteria thereby halting growthMonday, September 3, 2012
  • 106. TYPES OF INHIBITION: CompetitiveMonday, September 3, 2012
  • 107. TYPES OF INHIBITION: Non-CompetitiveMonday, September 3, 2012
  • 108. TYPES OF INHIBITION : Feedback InhibitionMonday, September 3, 2012
  • 109. START HEREMonday, September 3, 2012
  • 110. RIBOZYMES: molecular scissors • RNA enzymes: act on strands of RNA by removing sections and splicing together the remaining pieces • similarity with protein enzymes: function as catalysts, have active sites and are not used up in chemical reactions • difference with protein enzymes: more restricted substrate diversityMonday, September 3, 2012
  • 111. ENERGY PRODUCTION • Oxidation-Reduction (REDOX) reactions • Oxidation is the removal of electrons • Reduction is the gain of electrons • Redox reaction is an oxidation reaction paired with a reduction reactionMonday, September 3, 2012
  • 112. ENERGY PRODUCTION • Oxidation-Reduction (REDOX) reactions • In biological systems, the electrons are often associated with hydrogen atoms • Biological oxidations are often dehydrogenationsMonday, September 3, 2012
  • 113. ENERGY PRODUCTION • Generation of ATP : via phosphorylation of ADPMonday, September 3, 2012
  • 114. ENERGY PRODUCTION • 3 Ways of ATP Generation in Microbes • 1. Substrate-level Phosphorylation: ATP generated when a high-energy phosphate is directly transferred from a phosphorylated compound (substrate)Monday, September 3, 2012
  • 115. ENERGY PRODUCTION • 3 Ways of ATP Generation in Microbes • 2. Oxidative Phosphorylation: electrons are transferred from organic compounds to one group of electron carriers (NAD+ and FAD) via electron transport chain; ATP produced through (chemiosmosis) • 3. Photophosphorylation: occurs only in photosynthetic cells; Light causes chlorophyll to give up electrons; energy released from the transfer of electrons (oxidation) of chlorophyll through a system of carrier molecules is used to generate ATPMonday, September 3, 2012
  • 116. ENERGY PRODUCTIONMonday, September 3, 2012
  • 117. THE PATHWAYS OF ENERGY PRODUCTIONMonday, September 3, 2012
  • 118. HYPOTHETICAL PATHWAY OF ENERGY PRODUCTION • 1. Conversion of molecule A to B with reduction of NAD+ to NADH • 2. Conversion of molecule B to C • 3. Conversion of molecule C to D with conversion of ADP to ATP • 4. Irreversible conversion of D to E/E to D • 5. conversion of E to final product F using oxygen and producing carbon dioxide and waterMonday, September 3, 2012
  • 119. CARBOHYDRATE METABOLISMMonday, September 3, 2012
  • 120. • The breakdown of CARBOHYDRATE carbohydrates to release energy METABOLISM • Glycolysis: oxidation of glucose to pyruvic acid (with ATP and NADH) • Krebs cycle: oxidation of acetyl coA to carbon dioxide (with ATP, NADH and FADH2) • Electron transport chain: oxidation of NADH and FADH2 to Net gain of 2 ATP for each molecule generate ATP of glucose that is oxidizedMonday, September 3, 2012
  • 121. MICROBES ALTERNATIVE TO GLYCOLYSIS • PENTOSE PHOSPHATE PATHWAY (hexose monophosphate shunt) • operates simultaneously with glycolysis • breakdown of 5-C sugars and glucose • e.g. Bacillus subtilis, E. coli, Leuconostoc mesenteroides, Enterococcus faecalisMonday, September 3, 2012
  • 122. MICROBES ALTERNATIVE TO GLYCOLYSIS • PENTOSE PHOSPHATE PATHWAY (hexose monophosphate shunt) • produces important intermediate pentoses (used for biosynthesis of nucleotides, amino acids and glucose during photosynthesis) • important producer of NADPH • net gain of 1 molecule of ATP per molecule of glucose oxidizedMonday, September 3, 2012
  • 123. MICROBES ALTERNATIVE TO GLYCOLYSIS • ENTNER-DOUDOROFF PATHWAY • produces 1 molecule of ATP and 2 molecules of NADPH per molecule of glucose • bacteria have the enzymes to metabolize glucose without PPP and glycolysis via the EDP • e.g. Rhizobium, Pseudomonas, Agrobacterium, Enterococcus faecalis (NOTE: not found in Gram-positive bacteria)Monday, September 3, 2012
  • 124. SUMMARY OF GLYCOLYSIS & ALTERNATIVESMonday, September 3, 2012
  • 125. CELLULAR RESPIRATION • ATP-generating process (oxidative) • molecules are oxidized • operation of the “electron transport chain” • 2 types of respiration: • aerobic respiration (O2 final electron acceptor) • anaerobic respiration (inorganic molecule final electron acceptor)Monday, September 3, 2012
  • 126. AEROBIC RESPIRATION • Krebs Cycle • Electron Transport Chain/System (ETC/ETS)Monday, September 3, 2012
  • 127. AEROBIC RESPIRATION • Krebs Cycle • Electron Transport Chain/System (ETC/ ETS)Monday, September 3, 2012
  • 128. ATP GENERATION via CHEMIOSMOSISMonday, September 3, 2012
  • 129. ATP GENERATION via CHEMIOSMOSISMonday, September 3, 2012
  • 130. SUMMARY: AEROBIC RESPIRATIONMonday, September 3, 2012
  • 131. VENUES Pathway Eukaryote Prokaryote Glycolysis Cytoplasm Cytoplasm Intermediate step Cytoplasm Cytoplasm Krebs cycle Mitochondrial matrix Cytoplasm ETC Mitochondrial inner Plasma membrane membraneMonday, September 3, 2012
  • 132. ANAEROBIC RESPIRATION • final acceptor is an inorganic • e.g. other bacteria molecule • use carbonate from • e.g. Pseudomonas, Bacillus methane • use nitrate ion form • The total ATP yield is less than nitrite as final electron in aerobic respiration because acceptor only part of the Krebs cycle operates under anaerobic • e.g. Desulfovibrio conditions (microbes tend to grow more slowly) • use sulfate from hydrogen sulfide as final electron acceptorMonday, September 3, 2012
  • 133. FERMENTATION a. any process that releases energy from sugars or other organic molecules by oxidation = does not require O2, the Krebs cycle, or an electron transport chain = uses an organic molecule as the final electron acceptor b. Two ATP molecules are produced by substrate-level phosphorylation   c. Electrons removed from the substrate reduce NAD+ to NADHMonday, September 3, 2012
  • 134. TYPES OF FERMENTATION • lactic acid fermentation • pyruvic acid is reduced by NADH to lactic acid (lactic acid fermenters include Streptococcus and Lactobacillus) • Lactic acid can be fermented to propionic acid and CO2 by Propionibacterium freudenreichii (Swiss cheese) • alcohol fermentation • acetaldehyde is reduced by NADH to produce ethanol (alcohol fermenters include yeasts and bacteria) • Ethanol can be fermented to acetic acid (vinegar) by Acetobacter • Acetic acid can be fermented to methane by MethanosarcinaMonday, September 3, 2012
  • 135. TYPES OF FERMENTATION • Heterolactic fermenters • use the pentose phosphate pathway to produce lactic acid and ethanol (E. coli, Salmonella, Enterobacter) • Homolactic fermenters • produce only lactic acid (e.g. Streptococcus, Lactobacillus, Bacillus)Monday, September 3, 2012
  • 136. INDUSTRY ADVANTAGEMonday, September 3, 2012
  • 137. RESPIRATION vs FERMENTATIONMonday, September 3, 2012
  • 138. LIPID CATABOLISM • Lipases hydrolyze lipids into glycerol and fatty acids • Fatty acids and other hydrocarbons are catabolized by beta oxidation • Beta oxidation produces two carbon units that are linked to CoA to make acetyl-CoA • Catabolic products can be further broken down in glycolysis and the Krebs cycleMonday, September 3, 2012
  • 139. PROTEIN CATABOLISM • Before amino acids can be catabolized, they must be converted to various substances that enter the Krebs cycle or glycolysis • Transamination (transfer of NH2), decarboxylation (removal of COOH), and dehydrogenation (H2) reactions convert the amino acids to be catabolized into substances that enter the glycolytic pathway or Krebs cycleMonday, September 3, 2012
  • 140. SUMMARY: LIPID & PROTEIN CATABOLISMMonday, September 3, 2012
  • 141. METABOLISM as CLUE for BACTERIAL IDMonday, September 3, 2012
  • 142. PHOTOSYNTHESIS • conversion of light energy into chemical energy • the resulting chemical energy will be used to convert CO2 to a more reduced form of carbon, primarily sugars (carbon fixation) • e.g. Plants, Algae, Cyanobacteria: use water as hydrogen donor to release O2 • 6 CO + 12 H2O + light energy 2 C6H12O6 + 6H2O + 6O2 • e.g. Purple Sulfur Bacteria and Green Sulfur Bacteria: use H2S as hydrogen donor to produce sulfur granules • 6 CO + 12 H2S + light energy 2 C6H12O6 + 6H2O + 12SMonday, September 3, 2012
  • 143. LIGHT-DEPENDENT & LIGHT- INDEPENDENT REACTIONS • Light-Dependent: Photophosphorylation • ATP generation • Cyclic (e returns to chlorophyll) - • Non-cyclic (e used to reduce NADP+, and - electrons are returned to chlorophyll from H2O or H2S) • Light-Independent: Calvin-Benson Cycle • no light requirement • CO is fixed to synthesize sugars 2Monday, September 3, 2012
  • 144. LIGHT- DEPENDENT REACTIONSMonday, September 3, 2012
  • 145. CALVIN- BENSON CYCLEMonday, September 3, 2012
  • 146. PHOTOSYNTHESIS COMPAREDMonday, September 3, 2012
  • 147. WORTH MENTIONING • Halobacterium • uses bacteriorhodopsin (instead of chlorophyll) to generate electrons for a chemiosmotic proton pumpMonday, September 3, 2012
  • 148. SUMMARY OF ENERGY PRODUCTION MECHANISMSMonday, September 3, 2012
  • 149. NUTRITION GROUPS BASED ON METABOLISMMonday, September 3, 2012
  • 150. METABOLISM FOR ENERGY USEMonday, September 3, 2012
  • 151. POLYSACCHARIDE BIOSYNTHESIS Glycogen is formed from ADPG (ATP + glucose 6-phosphate = adenosine diphosphoglucose) in bacteria and from UDPG in animals (UTP + glucose 6-phosphate = uridine diphosphoglucose). UDPNAc is the starting material for the biosynthesis of peptidoglycan (UTP + fructose 6-phosphate = UDP-N-acetylglucosamine)Monday, September 3, 2012
  • 152. LIPID BIOSYNTHESIS Lipids are synthesized form fatty acid and glycerol. Glycerol is derived from dihydroxyacetone phosphate, and fatty acids are built from acetyl CoAMonday, September 3, 2012
  • 153. Amino acids are required for protein biosynthesis. All amino acids can be synthesized either directly or indirectly from intermediates of carbohydrate metabolism, particularly from the Krebs cycle. AMINO ACID & PROTEIN BIOSYNTHESIS Not all organisms can do this. Some require preformed amino acids.Monday, September 3, 2012
  • 154. PURINE & PYRIMIDINE BIOSYNTHESIS The sugars composing nucleotides are derived from either the pentose phosphate pathway or the Entner-Doudoroff pathway. Carbon and nitrogen atoms from certain amino acids (aspartic acid, glycine, glutamic acid) form the backbones of the purines and pyrimidines. Includes DNA, RNA, ATP, NAD, NADP, FMN, and FAD.Monday, September 3, 2012
  • 155. INTEGRATION • Anabolic and catabolic reactions are integrated through a group of common intermediates • Such integrated metabolic pathways are referred to as amphibolic pathwaysMonday, September 3, 2012
  • 156. INTERESTINGLY METAB...Monday, September 3, 2012
  • 157. CONTROL OF MICROBIAL GROWTHMonday, September 3, 2012
  • 158. WHY THE NEED TO CONTROL MICROBIAL • to destroy pathogens and prevent their transmission • to reduce and eliminate microorganisms responsible for the contamination of water, food and other important substancesMonday, September 3, 2012
  • 159. IMPORTANT TERMINOLOGIESMonday, September 3, 2012
  • 160. IMPORTANT TERMINOLOGIESMonday, September 3, 2012
  • 161. WHAT ARE ANTIMICROBIAL AGENTS?Monday, September 3, 2012
  • 162. PATTERN OF MICROBIAL DEATH • Analogous to population growth, population death is an exponential process: plotting the log (population) vs time will produce a straight-line plot • Bacterial populations die at a constant logarithmic rateMonday, September 3, 2012
  • 163. ASSESSMENT OF EFFECTIVENESS • Direct Assessment • Bacterial Killing Curves: Plot log %survival vs a measure of the sterilizing agentMonday, September 3, 2012
  • 164. ASSESSMENT OF EFFECTIVENESS • Direct Assessment • Time-Dose Relationship: The effect of the treatment depends both on concentration used and exposure time • In order to kill all the cells in a particular culture, one can hold the time constant and vary the dose or keep the dose constant and vary the time • e.g., A high dose for a short time will have the same effect as a low dose for a longer period of timeMonday, September 3, 2012
  • 165. ASSESSMENT OF EFFECTIVENESS • Direct Assessment • Death Point: Treatment dose necessary to sterilize the system in a given amount of time • Thermal death point = temperature necessary to sterilize a culture in 10 min. • Death Time: Time necessary to sterilize a system with a particular treatment • Thermal death time = time in min. necessary to sterilize the culture when a particular temperature is appliedMonday, September 3, 2012
  • 166. • Direct Assessment • Decimal Reduction Time (D- value): Exposure time at a given temperature needed to reduce the ASSESSMENT number of viable microbes by 90% OF (1 log) EFFECTIVENESS • Most precise way to characterize heat sterilization • Plot of log (number of viable cells) vs. time of heating (min) • Death rate increases with increasing temperature • z value in the change in temperature required to reduce the D value to 1/10 of its valueMonday, September 3, 2012
  • 167. ASSESSMENT OF EFFECTIVENESS • Indirect Assessment • Sterility Indicators: Use certain bacterial endospores • The most durable life forms known. e.g., Geobacillus stearothermophilus spores are capable of surviving 5 min in an autoclave (121°C; 15 psi)Monday, September 3, 2012
  • 168. FACTORS INFLUENCING EFFECTIVENESSMonday, September 3, 2012
  • 169. ACTION OF MICROBIAL CONTROL AGENTSMonday, September 3, 2012
  • 170. PHYSICAL METHODS OF MICROBIAL CONTROLMonday, September 3, 2012
  • 171. 1. HEAT • All microbes have a maximum and a minimum temperature for growth • Almost all macromolecules lose their structure (i.e., denature) and ability to function at very high temperatures • Moist or dry heatMonday, September 3, 2012
  • 172. 1. HEAT: Moist Heat • Moist heat is more effective than dry heat because • It penetrates cellular structures better • It facilitates unfolding of proteins, degrading DNA and disrupting membranes • It causes hydrogen bond rearrangementMonday, September 3, 2012
  • 173. 1. HEAT: Dry Heat • Dry heat is basically an oxidative process that denatures proteins and DNA disrupts membranesMonday, September 3, 2012
  • 174. WHICH TO CHOOSE: Dry over Wet • Too high a temperature may destroy a food product or render a medium useless • Wet heat may cause metal instruments to rust • The presence of certain compounds (e.g., protein, sugars and fats) may increase the resistance of cells to heat • Microbial death is more rapid at acidic pH • High concentrations of sugars, proteins and fats decrease heat penetration • Dry cells and endospores are more resistant than wet cells Hot-air Autoclave Equivalent treatments 170˚C, 2 hr 121˚C, 15 minMonday, September 3, 2012
  • 175. AUTOCLAVINGMonday, September 3, 2012
  • 176. CANNING • Uses heat under pressure to sterilize and hermetic sealing to drive out oxygenMonday, September 3, 2012
  • 177. PASTEURIZATION • used to kill harmful organisms in food or beverages and to prevent spoilage • does not sterilize (NOTE: sterilization would destroy desirable properties of many foods and beverages) • 2 Processes: • LTH (low-temperature-hold) or bulk pasteurization: 62.8°C for 30 min • HTST (high temperature-short time) or flash pasteurization: 71.7°C for 15s • This method is preferable for milk because it alters the taste less, kills heat resistant microbes more effectively and can be done on a continuous flow basesMonday, September 3, 2012
  • 178. UHT STERILIZATION • Ultra high temperature sterilization • Sterilizes food and other products • 141°C for 4 - 15 s • allows for a continuous flow systemMonday, September 3, 2012
  • 179. 2. LOW HEAT • Refrigeration (0 to 7°C) and freezing • Does not kill all microbes but inhibits growthMonday, September 3, 2012
  • 180. 3. RADIATIONMonday, September 3, 2012
  • 181. APPLICATIONS OF RADIATION • medical supplies and food industry (spices and fresh meat) • working cabinets • isolation roomsMonday, September 3, 2012
  • 182. 4/5. FILTRATION & DESICCATION • Filtration removes microbes • Desiccation prevents metabolismMonday, September 3, 2012
  • 183. CHEMICAL METHODS OF MICROBIAL CONTROLMonday, September 3, 2012
  • 184. CATEGORIES: Exterior or SurfacesMonday, September 3, 2012
  • 185. CATEGORIES: Exterior or SurfacesMonday, September 3, 2012
  • 186. • PHENOLICS • ALCOHOLS • HALOGENS • HEAVY METALS • QUATERNARY AMMONIUM COMPOUNDS (QUATS) • ALDEHYDES • STERILIZING GASESMonday, September 3, 2012
  • 187. Chemical agent Effectiveness against Endospores Mycobacteria Phenolics Poor Good Quats None None Chlorines Fair Fair Alcohols Poor Good Glutaraldehyde Fair GoodMonday, September 3, 2012
  • 188. PHENOLICS • First widely used antiseptic and disinfectant • Joseph Lister (1867): reduced the risk of infection during operations • Example: LYSOLR • Act by denaturing proteins and disrupting cell membranesMonday, September 3, 2012
  • 189. PHENOLICS • First widely used antiseptic and disinfectant • Joseph Lister (1867): reduced the risk of infection during operations • Example: LYSOLR ADVANTAGES: effective in the presence of • Act by denaturing organic material and remain active on surfaces long after application proteins and disrupting cell DISADVANTAGE: disagreeable odor and can cause skin irritation and in some instances membranes brain damage (hexachlorophene)Monday, September 3, 2012
  • 190. • Widely used disinfectant and antiseptics ALCOHOLS • Bactericidal and fungicidal but not sporicidal • May not destroy lipid- containing viruses • Example: ethanol and isopropanol (70-80% concentration) • Act by denaturing proteins and possibly by dissolving membrane lipids • 10-15 soaking in alcohol is sufficient to disinfect thermometers and small instrumentsMonday, September 3, 2012
  • 191. HALOGENS • Widely used disinfectant and antiseptics • e.g. Iodine: Kills by oxidizing cell constituents and iodinating cell proteins; Kill spores at high concentrations • e.g. Chlorine: Usually for water supply; Kills by oxidation of cellular materials and destruction of vegetative bacteria, fungi (NOTE: Will not kill spores) • Death within 30 minutes • DISADVANTAGE: a stain may be leftMonday, September 3, 2012
  • 192. HEAVY METALS • Mercury, Arsenic, Zinc, Copper • Used as germicides • How do they Kill: • Heavy metals combine with proteins, often with their sulfhydryl groups and inactivate them • May also precipitate cell proteinsMonday, September 3, 2012
  • 193. QUATS • DETERGENTS: Amphipathic (both polar and non- polar ends) • Kill by disrupting microbial membranes and denature proteins • ADVANTAGE: stable, non-toxic • DISADVANTAGE: inactivated by hard water Soap Degerming Acid-anionic detergents Sanitizing Quarternary ammonium compounds Bactericidal, Denature proteins, disrupt Cationic detergents plasma membraneMonday, September 3, 2012
  • 194. ALDEHYDES • FORMALDEHYDES:Very reactive molecules that combine with proteins and inactivate them • Sporicidal and can be used as sterilantsMonday, September 3, 2012
  • 195. ASSESSMENT OF DISINFECTANT EFFICACYMonday, September 3, 2012
  • 196. ASSESSMENT OF DISINFECTANT EFFICACYMonday, September 3, 2012
  • 197. ISSUE: ANTIBIOTICS & RESISTANCEMonday, September 3, 2012
  • 198. ISSUE: ANTIBIOTICS & RESISTANCEMonday, September 3, 2012
  • 199. END OF EXAM COVERAGEMonday, September 3, 2012