Biology 120 lectures for 2nd exam 2012 2012

MICROBIAL GROWTH

AY 2012-2013

Monday, September 3, 2012

DEFINITION OF MICROBIAL GROWTH

• NUMBER OF CELLS

• NOT CELL SIZE

• e.g. Growing microbes = increase in numbers, accumulating colonies


DEFINITION OF MICROBIAL GROWTH

• Note: for coenocytic organisms (multinucleate): growth = increased cell size


FOR YOU TO GROW....


HOW ABOUT THEM?


RECALL MICROBIAL NUTRITION

CARBON SOURCESCARBON SOURCES

Autotrophs CO2 sole or principal biosynthetic carbon source

Heterotrophs Reduced, preformed, organic molecules from other organisms

ENERGY SOURCESENERGY SOURCES

Phototrophs Light

Chemotrophs Oxidation of organic or inorganic compounds

HYDROGEN AND ELECTRON SOURCESHYDROGEN AND ELECTRON SOURCES

Lithotrophs Reduced inorganic molecules

Organotrophs Organic moleculesMonday, September 3, 2012


MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, HYDROGEN/ELECTRONS AND CARBON

REPRESENTATIVE MICROORGANISMS

PHOTOLITHOTROPHIC AUTOTROPHY

Light energyInorganic hydrogen/electron donorCO2 carbon source

AlgaePurple and green sulfur bacteriaBlue-green algae (cyanobacteria)

PHOTOORGANOTROPHIC HETEROTROPHY

Light energyOrganic hydrogen/electron donorOrganic carbon source (CO2 may also be used)

Purple non-sulfur bacteriaGreen non-sulfur bacteria



MAJOR NUTRITIONAL TYPES SOURCES OF ENERGY, HYDROGEN/ELECTRONS AND CARBON

REPRESENTATIVE MICROORGANISMS

CHEMOLITHOTROPHIC AUTOTROPHY

Chemical energy source (inorganic)Inorganic hydrogen/electron donorCO2 carbon source

Sulfur-oxidizing bacteriaHydrogen bacteriaNitrifying bacteriaIron bacteria

CHEMOORGANOTROPHIC HETEROTROPHY

Chemical energy source (organic)Organic hydrogen/electron donorOrganic carbon source

ProtozoaFungiMost non-photosynthetic bacteria


REQUIREMENTS FOR MICROBIAL GROWTH

•PHYSICAL REQUIREMENTS

• TEMPERATURE

• pH

• OSMOTIC PRESSURE

•CHEMICAL REQUIREMENTS

• CARBON

• NITROGEN, SULFUR & PHOSPHORUS

• TRACE ELEMENTS

• OXYGEN

• ORGANIC GROWTH FACTORS


• “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)

REQUIREMENTS FOR MICROBIAL GROWTH: TEMPERATURE



MINIMUM, OPTIMUM, MAXIMUM


• Psychrotrophs: grow between 0°C and 20-30°C; cause food spoilage

• Hyperthermophiles

: extreme temperatures (members of the archaea)



• RECALL: pH acidity or alkalinity of a solution

• acidophiles

• neutrophiles

• alkaliphiles

REQUIREMENTS FOR MICROBIAL GROWTH: pH


• 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 water

REQUIREMENTS FOR MICROBIAL GROWTH: OSMOTIC PRESSURE


REQUIREMENTS FOR MICROBIAL GROWTH: OSMOTIC PRESSURE


• one of the most important requirements for microbial groth

• structural backbone of living matter

• e.g. Chemoautotrophs (carbon dioxide) and Chemoheterotrophs (organic materials)

REQUIREMENTS FOR MICROBIAL GROWTH:

CARBON


• ACCESS: amino acids and proteins

• Most bacteria decompose proteins

• Some bacteria use NH4+

or NO3–

• A few bacteria use N2 in nitrogen fixation

REQUIREMENTS FOR MICROBIAL GROWTH: NITROGEN


• ACCESS: amino acids, thiamine and biotin

• Most bacteria decompose proteins

• Some bacteria use SO4

2– or H2S

REQUIREMENTS FOR MICROBIAL GROWTH: SULFUR


• ACCESS: In DNA, RNA, ATP and membranes

• PO43– is a

source of phosphorus

REQUIREMENTS FOR MICROBIAL GROWTH: NITROGEN, SULFUR

AND PHOSPHORUS


• iron, copper, molybdenum, zinc

• essential for the function of co-factors

REQUIREMENTS FOR MICROBIAL GROWTH: TRACE ELEMENTS


REQUIREMENTS FOR MICROBIAL GROWTH: TRACE ELEMENTS

• BIOTIN

• Carboxylation (Leuconostoc)

• CYANOCOBALAMIN or VIT B12

• Molecular rearrangements (Euglena)

• FOLIC ACID

• One-carbon metabolism (Enterococcus)

• PANTOTHENIC ACID

• Fatty acid metabolism (Proteus)

• PYRIDOXINE or VIT B6

• Transamination (Lactobacillus)

• NIACIN

• Precursor of NAD and NADP (Brucella)

• RIBOFLAVIN or VIT B2

• Precursor of FAD and FMN (Caulobacter)

• THIAMINE or VIT B1

• Aldehyde group transfer (Bacillus


• “microbes that use molecular oxygen produce more energy from nutrients than microbes that do not use oxygen”

REQUIREMENTS FOR MICROBIAL GROWTH: OXYGEN


• aerobic bacteria

• anaerobic bacteria

• microaerophilic bacteria



• Microbes can be harmed by toxic forms of oxygen

• singlet oxygen (1O2-): 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 radiation




• peroxide anion (O22-): toxic; active ingredient in hydrogen peroxide and benzoyl peroxide

• SOLUTION: catalase and peroxidase




• 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 peroxide



• 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 pyrimidines

REQUIREMENTS FOR MICROBIAL GROWTH: ORGANIC GROWTH

FACTORS


REVISED SCHEDULE

DATE ACTIVITY

August 28 (2 hour) Microbial Growth and Metabolism/Physiology

August 28-September 4 (4 hours)

Metabolism and Physiology and Microbial Control

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 3


CULTURE MEDIAMonday, September 3, 2012

• 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 microorganisms

CULTURE MEDIA


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, plates


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)


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 fungi


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 oxygen


ANAEROBIC CULTURE TECHNIQUES


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 microbes



•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 bacteria



• Goal: to detect the presence of specific microorganisms associated with disease or poor sanitation

• DIFFERENTIAL: distinguish colonies of desired organisms when grown together with others


TYPES OF CULTURE MEDIA: Differential Media


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

PURE CULTURE


PREPARING PURE CULTURE

• Julius Richard Petri (1887)

• Easy to use, stackable (saving space), requirement for plating methods


OBTAINING PURE CULTURES: Streak Plating


PURE VS MIXED CULTURE


CHARACTERIZING COLONIES


CULTURE PRESERVATION


WAYS TO PRESERVE YOUR CULTURE

•subculturing

• mineral oil overlay

• freezing as glycerol stocks

• liquid nitrogen storage

• lyophilization



• subculturing

•mineral oil overlay



• lyophilization



• subculturing


•freezing as glycerol stocks


• lyophilization



• subculturing



•liquid nitrogen storage

• lyophilization



• subculturing




•lyophilization


REVIVAL OF PRESERVED L-DRIED CULTURES

http://www.jcm.riken.jpMonday, September 3, 2012

http://www.jcm.riken.jp

http://www.jcm.riken.jp

GROWTH OF BACTERIAL CULTURES


BACTERIAL DIVISION


OTHER FORMS OF DIVISION BY OTHER MICROBES

Budding = Rhodopseudomonas

Chains of conidiospores carried externally at the tips of the filaments = Actinomycetes Fragmentation of

filaments = Actinomycetes


THE MATHEMATICS OF GROWTH


CELL DIVISION• Generation

time: time required for a microbial population to double

• g = mean generation time

• g = t/nMonday, September 3, 2012

GENERATION TIME

•g = t/n


SAMPLE...

•Given an initial density of 4 x 104

•After 2 hours the cell density became 1 x 106

•Compute for the generation time

•Solution: t = 2

•n = [ log (1 x 106) – log (4 x 104)]/ 0.301; n = 4.65

•Generation time = (t/n); 2/4.65 or 0.43 hours OR 25.8 minutes


GENERATION TIME

MICROORGANISM TEMPERATURE (°C) GENERATION TIME (hours)

Escherichia coli 40 0.35

Bacillus subtilis 40 0.43

Mycobacterium tuberculosis

37 12

Euglena gracilis 25 10.9

Giardia lamblia 37 18

Sacharomyces cerevisiae

30 2


THE GROWTH CURVE


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 period


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 divide


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 recover


SHORT LAG PHASE

• SHORT LAG PHASE (or even absent)

• Young, vigorously growing exponential phase culture is transferred to fresh medium of same composition


LONG LAG PHASE• LONG LAG PHASE

• Inoculum is from an old culture

• Inoculum is from a refrigerated source

• Inoculation into a chemically-different medium


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 moment


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 products


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 cells


DIRECT MEASUREMENT

• Plate counts

• Filtration

• Most Probable Number (MPN)

• Direct Microscopic Count


PLATE COUNTS


RECALL: HOW TO COMPUTE CFU


FILTRATION


MPN


DMC


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 molds


MICROBIAL METABOLISM & PHYSIOLOGY


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 ammonia


RECALLING THE BASICS


DEFINITION

• METABOLISM: The sum of the chemical reactions in an organism

• CATABOLISM: The energy-releasing processes

• ANABOLISM: The energy-using processes


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 heat


ENZYMESMonday, September 3, 2012

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 formed


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 energy: requires a specific level of energy

• their specific chemical configurations: no reaction will take place unless the particles are properly oriented toward each other




ACTIVATION ENERGY & REACTION RATES

• REACTION RATES: frequency of collisions containing sufficient energy to bring about a reaction

• ACTIVATION ENERGY: amount of energy needed to disrupt the stable electronic configuration of any specific molecule so that the electrons can be rearranged


ENZYMES & CHEMICAL REACTIONS

• Enzymes speed up chemical reactions (biological catalysts)


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)


• names will usually end in -ase and grouped according to type of chemical reaction they catalyze


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 A


MECHANISM OF ENZYME ACTION


FACTORS INFLUENCING ENZYME ACTIVITY



•Temperature

• pH

• Substrate concentration

• Inhibitors



• Temperature

• pH


• Inhibitors



• Temperature

• pH


• Inhibitors

Sulfanilamide as inhibitor of PABA during folate synthesis in bacteria

thereby halting growth


TYPES OF INHIBITION: Competitive


TYPES OF INHIBITION: Non-Competitive


TYPES OF INHIBITION: Feedback Inhibition


START HERE


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 diversity


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 reaction


ENERGY PRODUCTION

• Oxidation-Reduction (REDOX) reactions

• In biological systems, the electrons are often associated with hydrogen atoms

• Biological oxidations are often dehydrogenations


ENERGY PRODUCTION

• Generation of ATP : via phosphorylation of ADP


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)


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 ATP


ENERGY PRODUCTION


THE PATHWAYS OF ENERGY PRODUCTION


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 water


CARBOHYDRATE METABOLISM


CARBOHYDRATE METABOLISM

• The breakdown of carbohydrates to release energy

• 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 generate ATP

Net gain of 2 ATP for each molecule of glucose that is oxidized


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 faecalis



• 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 oxidized



• 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)


SUMMARY OF GLYCOLYSIS & ALTERNATIVES


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)


AEROBIC RESPIRATION

• Krebs Cycle

• Electron Transport Chain/System (ETC/ETS)


ATP GENERATION via CHEMIOSMOSIS


SUMMARY: AEROBIC RESPIRATION


VENUES

Pathway Eukaryote Prokaryote

Glycolysis Cytoplasm Cytoplasm

Intermediate step Cytoplasm Cytoplasm

Krebs cycle Mitochondrial matrix Cytoplasm

ETC Mitochondrial inner membrane

Plasma membrane


ANAEROBIC RESPIRATION

• final acceptor is an inorganic molecule

• e.g. Pseudomonas, Bacillus

• use nitrate ion form nitrite as final electron acceptor

• e.g. Desulfovibrio

• use sulfate from hydrogen sulfide as final electron acceptor

• e.g. other bacteria

• use carbonate from methane

• The total ATP yield is less than in aerobic respiration because only part of the Krebs cycle operates under anaerobic conditions (microbes tend to grow more slowly)


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 NADH


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 Methanosarcina


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)


INDUSTRY ADVANTAGE


RESPIRATION vs FERMENTATION


• 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 cycle

LIPID CATABOLISM


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 cycle


SUMMARY: LIPID & PROTEIN CATABOLISM


METABOLISM as CLUE for BACTERIAL ID


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 CO2 + 12 H2O + light energy C6H12O6 + 6H2O + 6O2

• e.g. Purple Sulfur Bacteria and Green Sulfur Bacteria: use H2S as hydrogen donor to produce sulfur granules

• 6 CO2 + 12 H2S + light energy C6H12O6 + 6H2O + 12S


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

• CO2 is fixed to synthesize sugarsMonday, September 3, 2012

LIGHT-DEPENDENT REACTIONS


CALVIN-BENSON CYCLE


PHOTOSYNTHESIS COMPARED


WORTH MENTIONING

• Halobacterium

• uses bacteriorhodopsin (instead of chlorophyll) to generate electrons for a chemiosmotic proton pump


SUMMARY OF ENERGY

PRODUCTION MECHANISMS


NUTRITION GROUPS BASED ON METABOLISM


METABOLISM FOR ENERGY USE


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)


LIPID BIOSYNTHESIS

Lipids are synthesized form fatty acid and glycerol. Glycerol is derived from dihydroxyacetone phosphate, and fatty acids are built from acetyl CoA


AMINO ACID & PROTEIN

BIOSYNTHESIS

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.

Not all organisms can do this. Some require preformed amino acids.


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.


INTEGRATION

• Anabolic and catabolic reactions are integrated through a group of common intermediates

• Such integrated metabolic pathways are referred to as amphibolic pathways


INTERESTINGLY METAB...


CONTROL OF MICROBIAL GROWTH


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 substances


IMPORTANT TERMINOLOGIES


WHAT ARE ANTIMICROBIAL AGENTS?


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 rate


ASSESSMENT OF EFFECTIVENESS

• Direct Assessment

• Bacterial Killing Curves: Plot log %survival vs a measure of the sterilizing agent




• 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 time




• 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 applied


ASSESSMENT OF

EFFECTIVENESS


• Decimal Reduction Time (D-value): Exposure time at a given temperature needed to reduce the number of viable microbes by 90% (1 log)

• 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 value



• 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)


FACTORS INFLUENCING EFFECTIVENESS


ACTION OF MICROBIAL CONTROL AGENTS


PHYSICAL METHODS OF MICROBIAL CONTROL


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 heat


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 rearrangement


1. HEAT: Dry Heat

• Dry heat is basically an oxidative process that denatures proteins and DNA disrupts membranes


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

AUTOCLAVING


CANNING

• Uses heat under pressure to sterilize and hermetic sealing to drive out oxygen


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 bases


UHT STERILIZATION

• Ultra high temperature sterilization

• Sterilizes food and other products

• 141°C for 4 - 15 s

• allows for a continuous flow system


2. LOW HEAT

• Refrigeration (0 to 7°C) and freezing

• Does not kill all microbes but inhibits growth


3. RADIATION


APPLICATIONS OF RADIATION

• medical supplies and food industry (spices and fresh meat)

• working cabinets

• isolation rooms


4/5. FILTRATION & DESICCATION

• Filtration removes microbes

• Desiccation prevents metabolism


CHEMICAL METHODS OF MICROBIAL CONTROL


CATEGORIES: Exterior or Surfaces


• PHENOLICS

• ALCOHOLS

• HALOGENS

• HEAVY METALS

• QUATERNARY AMMONIUM COMPOUNDS (QUATS)

• ALDEHYDES

• STERILIZING GASES


Chemical agent Effectiveness againstEffectiveness againstEndospores Mycobacteria

Phenolics Poor GoodQuats None NoneChlorines Fair FairAlcohols Poor GoodGlutaraldehyde Fair Good


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 membranes


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 membranes

ADVANTAGES: effective in the presence of organic material and remain active on surfaces long after application

DISADVANTAGE: disagreeable odor and can cause skin irritation and in some instances brain damage (hexachlorophene)


ALCOHOLS• Widely used disinfectant and antiseptics

• 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 instruments


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 left


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 proteins


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 compoundsCationic detergents

Bactericidal, Denature proteins, disrupt plasma membrane


ALDEHYDES• FORMALDEHYDES: Very reactive molecules that

combine with proteins and inactivate them

• Sporicidal and can be used as sterilants


ASSESSMENT OF DISINFECTANT EFFICACY


ISSUE: ANTIBIOTICS & RESISTANCE


END OF EXAM COVERAGE


Biology 120 lectures for 2nd exam 2012 2012

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Transcript of Biology 120 lectures for 2nd exam 2012 2012