Chapter 5 Lecture Outline Environmental Influences and Control of Microbial Growth

Environmental Limits on Microbial Growth Temperature pH Osmolarity Oxygen Pressure Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. -phile means “MUST have” and not “likes it” Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Changes in Temperature Microbes cannot control their own temperature Growth rate increases with temperature Proteins denature if temperature too high Microbial proteins adapted to temperature range: Psychrophiles Cold: below 20°C Mesophiles 12°C–45°C Thermophiles 40°C–80°C Extreme thermophiles Hyperthermophile 65°C–113°C Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Psychrophile Environment Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Example for a Psychrophile Microorganism Halorubrum lacusprofundi Extremophile Haloarchaeon Found in subzero temperatures in 5 M NaCl Proteins more flexible and need less energy to function High content of unsaturated fatty acids makes membrane more fluid Model system for astrobiology Want to know more about astrobiology? http://www.atomseek.com/Astronomy/Extraterrestrial_Life/Exobiology/Geobiology_and_Astrobiology_at_Caltech/JPL_L90130 Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Thermophilic and Hyperthermophilic Environments Yellow Stone National Park: hot spring Hydrothermal vent in deep ocean Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Example for a Hyperthermophile Microroganism Thermus aquaticus First source for DNA polymerase used fro PCR amplification of DNA Stable enzymes due to decreased glycine content Stabilizing DNA binding proteins More saturated fatty acids in membrane Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Response to Temperature Heat shock response Occurs at high end of temperature range “Emergency” proteins produced Help keep proteins from denaturing Induced by many stressful conditions Heat High salt concentrations Arid conditions Cold shock response Response Analysis to Stress Red: genes up in condition 1 only Green: genes up in condition 2 only Orange-yellow: no change in gene expression Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Variations in Pressure Barophiles Adapted to high pressures Up to 1,000 atm Barotolerant organisms Grow at high, but not very high pressure Barosensitive organisms Die at high pressure Most “typical” bacteria, all mammals Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Barophilic Environment Example: Shewanella violacea Either psychrophile or hyperthermophile High levels of polyunsaturated fatty acids Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Changes in Water Activity and Salt Concentration Water activity relates to water availability Solutes raise osmolarity High osmolarity reduces available water or water activity Osmotic pressure can burst membranes Low osmotic pressure outside cell Mechanosensitive channels relieve stress Release cell contents High osmotic pressure outside cell Cells synthesize osmolytes to counter balance Proline, glutamic acid Increase internal osmolarity Global response Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Halophiles Archaea Require high concentration of NaCl 2–4 M (= 10 - 20% NaCl) sea water: contains ~ 3.5% NaCl Intracellular salt level is NOT high Pump out salt Na+/K+ antiport Live in salt seas Often reddish pigments Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Changes in pH Enzymes only work in narrow pH range Amino acids must have correct charges pH levels alter concentration of H+ Bacteria regulate internal pH When environment is in similar pH range Weak acids can pass through membranes Disrupt cell pH homeostasis, kills cells Good food preservatives Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Classification of Organisms Grouped by Optimal Growth pH Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. pH Homeostasis Acid added at a constant rate Base added at a constant rate Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Changes in pH Neutralophiles Acidophiles Alkalophiles Grow at pH 5–8 Include bacteria in gut Acidophiles Grow at pH 0–5 Some grow in stomach acid Some in sulfuric acid springs Alkalophiles Grow at pH 9–11 Found in soda lakes Sulfolobulus acidocaldarius 60 – 95°C, pH 1-5 Sulfur Caldron acid spring Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

pH Change can Trigger Up-Regulation of Virulence factor Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbial Responses to Oxygen Has microbe the machinery that allows to deal with oxygen radials? Terminal electron acceptor is oxygen. Respiration Terminal electron acceptor is a an organic endogenous compound fermentation Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Oxygen as Electron Acceptor Aerobes–O2 is ultimate electron acceptor Very strong electron acceptor Can oxidize, damage proteins Anaerobes Reactive oxygen species (ROS) produced Oxidize, damage proteins Microaerophiles Can tolerate low levels of O2 Catalase inactivates ROS Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Other Electron Acceptors Anaerobes pass electrons to different ultimate electron acceptors Anaerobic respiration Inorganic electron acceptors Nitrate nitrite, thiosulfate Fermentation Organic electron acceptors Thrive in anaerobic environments Early Earth, deep water, lower gut Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Oxygen-related Growth Zones Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Anaerobic Growth Technology Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Nutrient Deprivation, Starvation Starving E. coli Lack of nutrients slows cell metabolism Accumulation of smallsignaling molecules (e.g., cAMP) Global change in gene expression Stimulates stress responses Daughter cells become smaller Cells spread farther Sporulation Oligotrophs Most microbes in nature Efficiently absorb N2, PO4 from nutrient-poor (low in organic material) environments May commit suicide in high nutrient environments Starving Pseudomonas Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Maintaining Microbial Diversity through Nutrient Limitation A transports nitrogen poorly but phosphate efficiently B transports phosphate poorly but nitrogen efficiently A and B have enough phosphate but B has still advantage of better nitrogen uptake. Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Control of Microbial Growth Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Antimicrobial Growth Measures Sterilization ALL living cells including spores, dormant forms, and viruses eliminated Disinfection Killing or removal of DISEASE PRODUCING organisms (on non-animated surfaces) Antisepsis Killing or removal of DISEASE PRODUCING organisms on animated surfaces Sanitation Reducing the microbial population to a SAFE LEVEL for the public Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Control of Microbial Growth Physical Temperature Cold Heat Pasteurization Sterilzation Pressure Drying (lyophilization) Filtration Irradiation Chemical Biological Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Controlling Microbial Growth Microbes die at logarithmic rate D-value = Decimal reduction time D-value = time to kill 90% of cells 2 D-values = time to kill 99% of cells Antimicrobial agents decrease D-value Kills cells faster Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Physical Measures to Control Microbial Growth Temperature Heat Pasteurization Sterilization Cold Pressure Drying (lyophilization) Filtration Irradiation Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Physical Agents: Pasteurization Heating food to remove disease causing and reduces spoilage microorganisms Lead organisms are Mycobacterium tuberculosis and Coxiella burnetti Low Temperature Long Time 63°C for 30 minutes High Temperature Short Time (Flash pasteurization) 72°C for 15 seconds Pasteurization treatments do NOT kill all cells Pasteurized food spoils eventually Leaves food tasting normal UHT—Ultra-high temperature 150°C for 3 seconds Used for creamers Does not sterilize Sterilizes Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Physical Agents: Autoclave Moist heat more effective than dry heat Autoclave = steam cooker High temperature under pressure 121°C, 15 psi (2 atm) for 20 minutes Sterilizes Kills all bacteria Kills endospores Clostridium botulinum Botulism Bacillus anthracis Anthrax Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Physical Agents: Dry Heat 2 – 3 hrs 160 – 170° C Sterilization Prevents corrosion Suited also for powders Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Physical Agents: Cold Refrigeration (4 – 8°C) Slows growth, does not kill all bacteria Not: microorganisms die slower too! Most pathogens are mesophilic Exception: Listeria monocyogenes Freezing Kills some worms Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Physical Agents: Filtration 0. 2 mm Removes most bacteria Does not remove cell wall less bacteria Does not remove viruses Used for liquids and air May remove solutes that adhere to the filters Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Physical Agents: Irradiation Deinococcus radiodurans Would survive atomic bomb Extensive DNA repair mechanisms Used in bioremediation DNA damage UV Only for surface sterilization X-rays g-rays Electron beams Not suited for viruses and prions Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Chemical Agents Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Chemical Agents: Factors Influencing Efficacy Presence of organic matter (as less as better) Composition of microbial load Chemical stability of the agent Surface tension (should be low) Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Chemical Agents: Disinfectants Liquid Gaseous Kill microbes but destroys eukaryotic cells as well Cannot be used inside patients Example: halogens Iodine, chlorine Highly reactive Damage protein, lipids and DNA Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Chemical Structures and Microbial Targets of Selected Disinfectants Disruption of membranes Protein denaturation Proteins Lipids Proteins Nucleic acids Membranes Protein denaturation Protein Rapid penetration Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Chemical Agents Antibiotics Will be covered later Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Biological Agents Probiotics “Live bacteria ingested to the benefit of health Displace disease organisms from tissues Bacteriophages “Phages” Viruses that attack bacteria Do not harm eukaryotes Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz Microbes that grow at temperatures between 40°C and 80°C are called psychrophiles. mesophiles. thermophiles. extreme thermophiles. Answer: C Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz Bacteria cannot grow in solutions with very high concentrations of sugar because bacteria cannot digest pure sugar. sugar raises the solution’s osmolarity. sugar alters the solution’s pH. Answer: B Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc. Concept Quiz Physical agents to prevent bacterial growth include pasteurization, freezing, phages. irradiation, probiotics, filtration. autoclaving, irradiation, freezing. antibiotics, refrigeration, pasteurization. Answer: C Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.