1. Chapter 5 Lecture Outline
Environmental Influences and Control of Microbial Growth

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

3. 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.

4. 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
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5. Psychrophile Environment
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

6. 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?
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7. Thermophilic and Hyperthermophilic Environments
Yellow Stone National Park: hot spring
Hydrothermal vent in deep ocean
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8. 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
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9. 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.

10. 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.

11. Barophilic Environment
Example: Shewanella violacea
Either psychrophile or hyperthermophile
High levels of polyunsaturated fatty acids
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12. 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
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Halophiles
Archaea
Require high concentration of NaCl
2–4 M (= % 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
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14. 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.

15. Classification of Organisms Grouped by Optimal Growth pH
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pH Homeostasis
Acid added at a constant rate
Base added at a constant rate
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

17. 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.

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

19. 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.

20. 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.

21. 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
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23. Oxygen-related Growth Zones
Microbiology: An Evolving Science © 2009 W. W. Norton & Company, Inc.

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

25. 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.

26. 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.

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

28. 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.

29. 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.

30. 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.

31. 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.

32. 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.

33. 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.

34. Physical Agents: Dry Heat
2 – 3 hrs 160 – 170° C
Sterilization
Prevents corrosion
Suited also for powders
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35. 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.

36. 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.

37. 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.

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

39. 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.

40. 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.

41. Chemical Structures and Microbial Targets of Selected Disinfectants
Disruption of membranes
Protein
denaturation
Proteins
Lipids
Proteins
Nucleic acids
Membranes
Protein denaturation
Protein
Rapid penetration
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42. 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.

43. 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.

44. 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.

45. 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.

46. 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.