Biology 120 lectures for 2nd exam 2012 2012

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Biology 120 lectures for 2nd exam 2012 2012

  1. 1. MICROBIAL GROWTH AY 2012-2013Monday, September 3, 2012
  2. 2. DEFINITION OF MICROBIAL GROWTH • NUMBER OF CELLS • NOT CELL SIZE • e.g. Growing microbes = increase in numbers, accumulating coloniesMonday, September 3, 2012
  3. 3. DEFINITION OF MICROBIAL GROWTH • Note: for coenocytic organisms (multinucleate): growth = increased cell sizeMonday, September 3, 2012
  4. 4. FOR YOU TO GROW....Monday, September 3, 2012
  5. 5. HOW ABOUT THEM?Monday, September 3, 2012
  6. 6. HOW ABOUT THEM?Monday, September 3, 2012
  7. 7. HOW ABOUT THEM?Monday, September 3, 2012
  8. 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. 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. 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. 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. 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. 13. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATURE MINIMUM, OPTIMUM, MAXIMUMMonday, September 3, 2012
  14. 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. 15. REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATUREMonday, September 3, 2012
  16. 16. REQUIREMENTS FOR MICROBIAL GROWTH: pH • RECALL: pH acidity or alkalinity of a solution • acidophiles • neutrophiles • alkaliphilesMonday, September 3, 2012
  17. 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. 18. REQUIREMENTS FOR MICROBIAL GROWTH: OSMOTIC PRESSUREMonday, September 3, 2012
  19. 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. 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. 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. 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. 23. REQUIREMENTS FOR MICROBIAL GROWTH: TRACE ELEMENTS • iron, copper, molybdenum, zinc • essential for the function of co- factorsMonday, September 3, 2012
  24. 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. 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. 26. Monday, September 3, 2012
  27. 27. REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN • aerobic bacteria • anaerobic bacteria • microaerophilic bacteriaMonday, September 3, 2012
  28. 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. 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. 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. 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. 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. 33. CULTURE MEDIAMonday, September 3, 2012
  34. 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. 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. 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. 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. 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. 39. ANAEROBIC CULTURE TECHNIQUESMonday, September 3, 2012
  40. 40. ANAEROBIC CULTURE TECHNIQUESMonday, September 3, 2012
  41. 41. ANAEROBIC CULTURE TECHNIQUESMonday, September 3, 2012
  42. 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. 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. 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. 45. TYPES OF CULTURE MEDIA: Differential MediaMonday, September 3, 2012
  46. 46. TYPES OF CULTURE MEDIA: Differential MediaMonday, September 3, 2012
  47. 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. 48. PURE CULTUREMonday, September 3, 2012
  49. 49. PREPARING PURE CULTURE • Julius Richard Petri (1887) • Easy to use, stackable (saving space), requirement for plating methodsMonday, September 3, 2012
  50. 50. OBTAINING PURE CULTURES: Streak PlatingMonday, September 3, 2012
  51. 51. PURE VS MIXED CULTUREMonday, September 3, 2012
  52. 52. CHARACTERIZING COLONIESMonday, September 3, 2012
  53. 53. CULTURE PRESERVATIONMonday, September 3, 2012
  54. 54. WAYS TO PRESERVE YOUR CULTURE •subculturing • mineral oil overlay • freezing as glycerol stocks • liquid nitrogen storage • lyophilizationMonday, September 3, 2012
  55. 55. WAYS TO PRESERVE YOUR CULTURE • subculturing •mineral oil overlay • freezing as glycerol stocks • liquid nitrogen storage • lyophilizationMonday, September 3, 2012
  56. 56. WAYS TO PRESERVE YOUR CULTURE • subculturing • mineral oil overlay •freezing as glycerol stocks • liquid nitrogen storage • lyophilizationMonday, September 3, 2012
  57. 57. WAYS TO PRESERVE YOUR CULTURE • subculturing • mineral oil overlay • freezing as glycerol stocks •liquid nitrogen storage • lyophilizationMonday, September 3, 2012
  58. 58. WAYS TO PRESERVE YOUR CULTURE • subculturing • mineral oil overlay • freezing as glycerol stocks • liquid nitrogen storage •lyophilizationMonday, September 3, 2012
  59. 59. REVIVAL OF PRESERVED L- DRIED CULTURES http://www.jcm.riken.jpMonday, September 3, 2012
  60. 60. GROWTH OF BACTERIAL CULTURESMonday, September 3, 2012
  61. 61. BACTERIAL DIVISIONMonday, September 3, 2012
  62. 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. 63. THE MATHEMATICS OF GROWTHMonday, September 3, 2012
  64. 64. CELL DIVISION • Generation time: time required for a microbial population to double • g = mean generation time • g = t/nMonday, September 3, 2012
  65. 65. GENERATION TIME • g = t/nMonday, September 3, 2012
  66. 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. 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. 68. THE GROWTH CURVEMonday, September 3, 2012
  69. 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. 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. 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. 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. 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. 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. 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. 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. 77. DIRECT MEASUREMENT • Plate counts • Filtration • Most Probable Number (MPN) • Direct Microscopic CountMonday, September 3, 2012
  78. 78. PLATE COUNTSMonday, September 3, 2012
  79. 79. RECALL: HOW TO COMPUTE CFUMonday, September 3, 2012
  80. 80. FILTRATIONMonday, September 3, 2012
  81. 81. MPNMonday, September 3, 2012
  82. 82. DMCMonday, September 3, 2012
  83. 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. 84. MICROBIAL METABOLISM & PHYSIOLOGYMonday, September 3, 2012
  85. 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. 86. RECALLING THE BASICSMonday, September 3, 2012
  87. 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. 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. 89. ENZYMESMonday, September 3, 2012
  90. 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. 91. FACTORS THAT DETERMINE WHETHER A COLLISION WILL CAUSE A CHEMICAL REACTIONMonday, September 3, 2012
  92. 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. 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. 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. 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. 96. ENZYMES & CHEMICAL REACTIONS • Enzymes speed up chemical reactions (biological catalysts)Monday, September 3, 2012
  97. 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. 98. • names will usually end in -ase and grouped according to type of chemical reaction they catalyzeMonday, September 3, 2012
  99. 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. 100. MECHANISM OF ENZYME ACTIONMonday, September 3, 2012
  101. 101. FACTORS INFLUENCING ENZYME ACTIVITYMonday, September 3, 2012
  102. 102. FACTORS INFLUENCING ENZYME ACTIVITY • Temperature • pH • Substrate concentration • InhibitorsMonday, September 3, 2012
  103. 103. FACTORS INFLUENCING ENZYME ACTIVITY • Temperature • pH • Substrate concentration • InhibitorsMonday, September 3, 2012
  104. 104. FACTORS INFLUENCING ENZYME ACTIVITY • Temperature • pH • Substrate concentration • InhibitorsMonday, September 3, 2012
  105. 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. 106. TYPES OF INHIBITION: CompetitiveMonday, September 3, 2012
  107. 107. TYPES OF INHIBITION: Non-CompetitiveMonday, September 3, 2012
  108. 108. TYPES OF INHIBITION : Feedback InhibitionMonday, September 3, 2012
  109. 109. START HEREMonday, September 3, 2012
  110. 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. 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. 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. 113. ENERGY PRODUCTION • Generation of ATP : via phosphorylation of ADPMonday, September 3, 2012
  114. 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. 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. 116. ENERGY PRODUCTIONMonday, September 3, 2012
  117. 117. THE PATHWAYS OF ENERGY PRODUCTIONMonday, September 3, 2012
  118. 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. 119. CARBOHYDRATE METABOLISMMonday, September 3, 2012
  120. 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. 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. 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. 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. 124. SUMMARY OF GLYCOLYSIS & ALTERNATIVESMonday, September 3, 2012
  125. 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. 126. AEROBIC RESPIRATION • Krebs Cycle • Electron Transport Chain/System (ETC/ETS)Monday, September 3, 2012
  127. 127. AEROBIC RESPIRATION • Krebs Cycle • Electron Transport Chain/System (ETC/ ETS)Monday, September 3, 2012
  128. 128. ATP GENERATION via CHEMIOSMOSISMonday, September 3, 2012
  129. 129. ATP GENERATION via CHEMIOSMOSISMonday, September 3, 2012
  130. 130. SUMMARY: AEROBIC RESPIRATIONMonday, September 3, 2012
  131. 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. 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. 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. 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. 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. 136. INDUSTRY ADVANTAGEMonday, September 3, 2012
  137. 137. RESPIRATION vs FERMENTATIONMonday, September 3, 2012
  138. 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. 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. 140. SUMMARY: LIPID & PROTEIN CATABOLISMMonday, September 3, 2012
  141. 141. METABOLISM as CLUE for BACTERIAL IDMonday, September 3, 2012
  142. 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. 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. 144. LIGHT- DEPENDENT REACTIONSMonday, September 3, 2012
  145. 145. CALVIN- BENSON CYCLEMonday, September 3, 2012
  146. 146. PHOTOSYNTHESIS COMPAREDMonday, September 3, 2012
  147. 147. WORTH MENTIONING • Halobacterium • uses bacteriorhodopsin (instead of chlorophyll) to generate electrons for a chemiosmotic proton pumpMonday, September 3, 2012
  148. 148. SUMMARY OF ENERGY PRODUCTION MECHANISMSMonday, September 3, 2012
  149. 149. NUTRITION GROUPS BASED ON METABOLISMMonday, September 3, 2012
  150. 150. METABOLISM FOR ENERGY USEMonday, September 3, 2012
  151. 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. 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. 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. 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. 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. 156. INTERESTINGLY METAB...Monday, September 3, 2012
  157. 157. CONTROL OF MICROBIAL GROWTHMonday, September 3, 2012
  158. 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. 159. IMPORTANT TERMINOLOGIESMonday, September 3, 2012
  160. 160. IMPORTANT TERMINOLOGIESMonday, September 3, 2012
  161. 161. WHAT ARE ANTIMICROBIAL AGENTS?Monday, September 3, 2012
  162. 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. 163. ASSESSMENT OF EFFECTIVENESS • Direct Assessment • Bacterial Killing Curves: Plot log %survival vs a measure of the sterilizing agentMonday, September 3, 2012
  164. 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. 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. 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. 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. 168. FACTORS INFLUENCING EFFECTIVENESSMonday, September 3, 2012
  169. 169. ACTION OF MICROBIAL CONTROL AGENTSMonday, September 3, 2012
  170. 170. PHYSICAL METHODS OF MICROBIAL CONTROLMonday, September 3, 2012
  171. 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. 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. 173. 1. HEAT: Dry Heat • Dry heat is basically an oxidative process that denatures proteins and DNA disrupts membranesMonday, September 3, 2012
  174. 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. 175. AUTOCLAVINGMonday, September 3, 2012
  176. 176. CANNING • Uses heat under pressure to sterilize and hermetic sealing to drive out oxygenMonday, September 3, 2012
  177. 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. 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. 179. 2. LOW HEAT • Refrigeration (0 to 7°C) and freezing • Does not kill all microbes but inhibits growthMonday, September 3, 2012
  180. 180. 3. RADIATIONMonday, September 3, 2012
  181. 181. APPLICATIONS OF RADIATION • medical supplies and food industry (spices and fresh meat) • working cabinets • isolation roomsMonday, September 3, 2012
  182. 182. 4/5. FILTRATION & DESICCATION • Filtration removes microbes • Desiccation prevents metabolismMonday, September 3, 2012
  183. 183. CHEMICAL METHODS OF MICROBIAL CONTROLMonday, September 3, 2012
  184. 184. CATEGORIES: Exterior or SurfacesMonday, September 3, 2012
  185. 185. CATEGORIES: Exterior or SurfacesMonday, September 3, 2012
  186. 186. • PHENOLICS • ALCOHOLS • HALOGENS • HEAVY METALS • QUATERNARY AMMONIUM COMPOUNDS (QUATS) • ALDEHYDES • STERILIZING GASESMonday, September 3, 2012
  187. 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. 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. 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. 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. 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. 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. 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. 194. ALDEHYDES • FORMALDEHYDES:Very reactive molecules that combine with proteins and inactivate them • Sporicidal and can be used as sterilantsMonday, September 3, 2012
  195. 195. ASSESSMENT OF DISINFECTANT EFFICACYMonday, September 3, 2012
  196. 196. ASSESSMENT OF DISINFECTANT EFFICACYMonday, September 3, 2012
  197. 197. ISSUE: ANTIBIOTICS & RESISTANCEMonday, September 3, 2012
  198. 198. ISSUE: ANTIBIOTICS & RESISTANCEMonday, September 3, 2012
  199. 199. END OF EXAM COVERAGEMonday, September 3, 2012

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