Lecture 05 2024.pdf

BIOL371: Microbiology Lecture 5 – environmental effects on growth and control of microbial growth 1  Topics of today 1. Temperature effects on growth 2. Osmolarity, pH and oxygen on growth 3. Controlling microbial growth Materials covered:...

BIOL371: Microbiology Lecture 5 – environmental effects on growth and control of microbial growth 1  Topics of today 1. Temperature effects on growth 2. Osmolarity, pH and oxygen on growth 3. Controlling microbial growth Materials covered:  Chapter 4.11-4.19  Figures 4.20-4.22, 4.24-4.31, 4.33-35, 4.37-4.41  Tables 4.4, 4.5, 4.7, 4.9 2  1. Temperature effects on growth 1. Temperature classes of microorganisms 2. Microbial life in the cold 3. Microbial life at high temperature 3  Cardinal temperatures  Cardinal temperatures: minimum, optimum, and maximum temperatures at which an organism grows  Characteristics of any given microorganism  Differ dramatically between species  At optimum, all/most cellular processes are functioning at maximum rate 4  Temperature classes of microorganisms  Four broad classes related to growth temperature optimum: psychrophile, mesophile (most commonly studied), thermophile, and hyperthermophile 5  Microbial life in the cold  Much of the Earth’s surface is cold  Oceans: ~5°C  Arctic and Antarctic are permanently frozen or rarely unfrozen  Constantly vs seasonally cold Edge of glacier and subglacial lakes Phase-contrast Transmission electron are teeming with microbial life Core of frozen micrograph of micrograph of gas- seawater phototrophic microbes vesiculated bacteria 6  Psychrophilic and psychrotolerant microorganisms  Psychrophiles: optimum at ≤ 15°C, maximum < 20°C, minimum ≤ 0°C  Constantly cold environments  Found in polar regions, permanent snowfields, glacier  Psychrotolerant: can grow at 0°C, but have optima of 20°C to 40°C  More widely distributed in nature than psychrophiles  Found in soils and water in temperate climates and food at 4°C 7  Molecular adaptations to life in the cold  Production of enzymes that function optimally in the cold  More alpha helices than beta-sheets, greater flexibility  More polar and fewer hydrophobic amino acids  Fewer weak bonds (e.g., hydrogen and ionic bonds)  Cytoplasmic membranes function at low temperatures  Higher content of unsaturated and shorter-chain fatty acid  Polyunsaturated fatty acids  Cold shock proteins (chaperones) in maintaining cold-sensitive proteins in active form  Cryoprotectants (e.g., antifreeze proteins, high concentration of solutes such as glycerol) prevent formation of ice crystals  Exopolysaccharide cell surface slime 8  Microbial life at high temperatures Boiling spring  Thermophiles: growth temperature optima between 45°C and 80°C  Hyperthermophiles: optima greater than 80°C  Inhabit hot environments; e.g., boiling hot springs and seafloor hydrothermal vents  Chemoorganotrophs and chemolithotrophs  Archaea and bacteria  Methanopyrus: most thermophilic of up to 122°C  Above 65°C, only prokaryotes can thrive; but extensive diversity 9 Upper temperature limits of growth of living organisms Group Upper temperature limits Aquatic animals 38°C Terrestrial animals 50°C Ostracods (crustaceans) 49–50°C Plants 45°C (60°C for one species) Eukaryotic microbes Protozoa 56°C Algae 60°C Fungi 62°C Bacteria Cyanobacteria 73°C Anoxygenic phototrophic bacteria 73°C Chemoorganotrophs/chemolithotrophs 95°C Archaea 122°C 10  Thermo-stable proteins  Enzymes and proteins more heat-stable and function optimally at high temperatures  Heat stability from subtle amino acid substitutions that resist denaturation  Increased ionic bonding and highly hydrophobic interiors  Production of solutes (e.g., di-inositol phosphate, diglycerol phosphate, mannosylglycerate) helps stabilize proteins.  Thermophilic and hyperthermophilic enzymes commercially useful  Prolong shelf life (e.g., Taq polymerase for polymerase chain reaction [PCR]) 11  Membrane stability at high temperatures  Cytoplasmic membranes must be heat stable to survive high temperatures  Bacteria have lipids rich in long-chain and saturated fatty acids, fewer unsaturated fatty acids  Hyperthermophiles of Archaea have C40 hydrocarbons made of repeating isoprene units bonded by ethers to glycerol phosphate, and membrane forms lipid monolayer rather than bilayer 12  Effects of pH on microbial growth  Most microbes have a pH range of 2-3 pH units within which growth is possible  Although some microorganisms can live at very low or very high pH, the cell’s internal pH remains near neutrality 13  Growth pH optima of microorganisms  Optimal pH for growth refers to extracellular pH only; intracellular pH must be maintained at near neutral pH, consistent with macromolecule stability  Neutrophiles: grow optimally at pH 5.5-7.9  Acidophiles: grow best at below pH 5.5  Alkaliphiles: grow optimally at above pH 8.0  Cytoplasmic membrane must remain stable at extreme pH  Some alkaliphiles use sodium motive force instead of proton motive force to generate energy  Culture media for microbes typically contain buffers to maintain constant pH  Extracellular enzymes from extremophiles are highly useful in industrial processes  Acidic pectinases used in fruit juice extraction  Alkaline proteases and lipases are used to remove fats and proteins in laundry detergents 14  Osmolarity and microbial growth  Water availability depends on environmental moisture/dryness and concentration of solutes  Water activity (aw): ratio of vapour pressure of air in equilibrium with a substance or solution to vapor pressure of pure water  Osmosis: water diffuses from high to low concentrations Water activity Material Example organisms 1.000 Pure water Caulobacter, Spirillum 0.995 Human blood Streptococcus, Escherichia 0.950 Bread Most gram-positive rods 0.900 Maple syrup, ham Gram-positive cocci such as Staphylococcus 0.750 Salt lakes, salted fish Halobacterium, Halococcus 0.700 Cereals, candy, dried fruit Xeromyces bisporus and other xerophilic fungi 15  Halophiles  Typically, the cytoplasm has a higher solute concentration than the surrounding environment; thus, tendency for water to move into the cell  When a cell is in an environment with a higher external solute concentration, water will flow out unless the cell has a mechanism to prevent this  Seawater contains ~3% NaCl  Halophiles have a specific requirement for ~3-6% NaCl  Halotolerant: tolerate some dissolved solutes but generally grow best in the absence of added solute  Extreme halophiles: require very high levels (15- 30%) of NaCl; often unable to grow at lower concentrations 16  Maintaining water balance  Halophiles and Related Organisms  Osmophiles: live in environments high in sugar  Xerophiles: able to grow in very dry environments  Compatible Solutes  Solutes bind water  To maintain water balance, microbes pump solutes from environment into cell or synthesizing cytoplasmic solutes  compatible solutes do not inhibit biochemical processes, highly water- soluble (e.g., sugars, alcohols, glycine betaine, KCl)  Nonhalotolerant, halotolerant, halophilic, extremely halophilic organisms reflect genetic ability to produce or accumulate compatible solutes 17  Oxygen classes of microorganisms  Most eukaryotes are obligate aerobes; facultative aerobes (e.g., yeast) and obligate anaerobes (e.g., ruminant fungi and some protozoans) are known Group Relationship to O2 Metabolism Example Habitat Obligate aerobes Required Aerobic respiration Micrococcus luteus Skin, dust Aerobic respiration, Not required, growth Mammalian large Facultative aerobes anaerobic respiration, Escherichia coli better with O2 intestine fermentation Required at level below Microaerophilic Aerobic respiration Spirillum volutans Lake water atmospheric O2 Not required, growth no Streptococcus Aerotolerant Fermentation Oral cavity better when is O2 present mutans Anaerobic respiration, Methanobacterium Obligate anaerobes Harmful or lethal Sewage sludge fermentation formicicum 18  Culturing aerobes and anaerobes  Aerobes need extensive aeration (e.g., shaking, bubbling)  Anaerobes need oxygen excluded  Reducing agents added to culture media to reduce oxygen to water (e.g., thioglycolate)  Oxygen can penetrate only the top of the tube  Grow in anaerobic chambers (e.g., glove box) can flush oxygen (a) Aerobic (b) Anaerobic (c) Facultative (d) Microaerophilic (e) Aerotolerant Anoxic glove box 19  Why is oxygen toxic?  Molecular oxygen is not toxic  By-products of the reduction of O2 to H2O are toxic  All organisms, when exposed to oxygen, will encountered toxic forms of oxygen  toxic oxygen species need to be removed 20  Enzymes that destroy toxic oxygen species Catalase H2 O2  H2 O2 2 H2 O  O2 Peroxidase H2 O2  NADH + H+ 2 H2 O  NAD Superoxide dismutase O2   O2   2 H H2 O2  O2 Superoxide reductase O2   2 H+  rubredoxinreduced H2 O2  rubredoxinoxidized 21  General principles of growth control  Decontamination: treatment of an object or surface to make it safe to handle  May simply involve wiping surface or washing to remove contaminants  Disinfection: directly targets pathogens, not necessary all microorganisms  Requires the use of agents called disinfectants  Kills or severely inhibits growth  Sterilization: killing of all microorganisms including viruses  General methods of controlling microbial growth:  Heat  Radiation  Filtration  Chemicals  Antimicrobials 22  Quantifying heat as a sterilant  Heat kills more rapidly as temperature increases  At a given temperature, moist heat kills faster than dry heat  Decimal reduction time: amount of time required at a given temperature to kill 90%; killing is exponential, thus expressed in logarithmic scale 10% survival A – mesophile B – thermophile C - hyperthermophile 23 Thermal death time as a measurement of heat sensitivity  Thermal death time: time to kill all cells at a given temperature  Affected by population size – longer time is required to kill a large population than a smaller population  Endospores can survive heat that would kill vegetative cells  Killing rate is affected by environmental conditions  Acidic pH kills faster – acidic foods such as tomatoes and pickles are easier to sterilize  Neural-pH foods such as beans and corn take longer time  High concentrations of sugars, proteins, and fats increase heat resistance of microbes 24  Autoclave and pasteurization  Autoclave: sealed heating device that uses steam under pressure yielding 121°C  High temperature, not pressure, that kills microbes  Kills endospores  Pasteurization: uses heat to reduce microbial load in heat-sensitive liquids  Kills all known pathogens  For milk: 15 seconds at 71°C (or 135°C for 1-2 seconds), then chill rapidly 25  Ultraviolet radiation  Ultraviolet (UV) radiation: between 220 and 300 nm  Affects DNA and causes mutations  Poor penetration power  Use to sterilize surface and air  Germicidal UV light used in laboratory biosafety hoods for sterilization between experiments 26  Ionizing radiation  Ionizing radiation: electromagnetic radiation that produces ions and other reactive molecules with which radiation particles collide  Generated by using X-ray or the radioactive nuclides cobalt-60 or cesium-137  Enough penetration power to kill microorganisms  Sensitivity to ionizing radiation:  Multicellular organisms more sensitive than microorganisms  Bacteria more sensitive than viruses  Growing cells more sensitive than endospores  Used in diverse applications: surgical instruments, plastic labware, fresh produce, meat etc 27  Filter sterilization  Membrane filters: fibrous sheets made of overlapping paper or glass fibres that traps particles  Pores of filters (0.2 and 0.45 μm) are too small for microbes to pass through  e.g., HEPA (high efficiency particulate air) filters remove particles (>0.3 μm in size) from air  Used on heat-sensitive liquid and gases  Nucleopore filters: thin irradiated film etched to make holes  Filtration units vary greatly in size; from a simple syringe to huge industrial scale Membrane filter Nucleopore filter 28  Antimicrobial agents  Antimicrobial agents: chemicals that kill or inhibit growth  -cidal: kills microorganisms; e.g., bactericidal, fungicidal, viricidal  -static: inhibits growth; e.g., bacteriostatic, fungistatic, viristatic  Antibacterial agents:  Bacteriostatic agents inhibit important biochemical processes  Bactericidal agents kill the cells without lysis  Bacteriolytic agents kill by lysis 29  Assaying antimicrobial activity: liquid assay  Minimum inhibitory concentration: smallest amount of an agent needed to inhibit growth of a microorganism  Growth on liquid media with dilutions of agents 30  Assaying antimicrobial activity: diffusion assay  Disk diffusion on solid media  Antimicrobial agent added to filter disk, diffuses to media  Zone of growth inhibition: area of no growth around disk 31  Examples of chemical antiseptics and sterilants  Antiseptics (germicides) kill or inhibit microbial growth and are nontoxic enough to use on living tissues  Sterilants destroy all microorganisms, including endospores Agent Mode of action Use Alcohol (60-85% ethanol or Lipid solvent and protein Topical antiseptic isopropanol in water) denaturant Hydrogen peroxide Oxidizing agent Topical antiseptic Iodophors (Betadine) Iodinate proteins Topical antiseptic Octenidine (cationic surfactant) Disrupts membrane Topical antiseptic Ethylene oxide (gas) Alkylating agent Sterilant Formaldehyde (37%) Alkylating agent Sterilant Hydrogen peroxide Oxidizing agent Vapor used as sterilant 32  Examples of chemical disinfectants and sanitizers  Disinfectants kill microorganisms but not necessarily endospores, use on surface  Sanitizers are less harsh, reduce microbial numbers, do not sterilize Agent Mode of action Use Alcohol (60-85% ethanol or Lipid solvent and protein General-purpose disinfectant for isopropanol in water) denaturant virtually all surfaces Chlorine gas Oxidizing agent Disinfectant for drinking water General-purpose disinfectant for Pine oil (Pine-Sol®) Protein denaturant household surface Ozone Strong oxidizing agent Disinfectant fro drinking water Alcohol (75%), hydrogen Lipid solvent and protein General-purpose sanitizer peroxide (0.1%), glycerol (1.5%) denaturant Copper sulfate Protein precipitant Algicide in swimming pool 33