2. Chapter Overview - Physical, chemical, and biological
● How the environment limits growth
● The microbial response to temperature
● How microbes cope with pressure
● The microbial response to changes in: water activity, salt concentrations, pH, and oxygen
● Hungry microbes
● The control of microbes:
- Physical, chemical, and biological

3. Introduction
Microbes have both the fastest and the slowest growth rates of known organisms
- Some hot-springs bacteria can double in as little as 10 minutes, whereas deep-seas sediment microbes may take as long as 100 years
These differences are determined by nutrition and niche-specific physical parameters like temperature and pH

4. Environmental Limits of Microbial Growth
“Normal” growth conditions
- Sea-level; temperature 20-40o C; neutral pH; 0.9% salt, and ample nutrients
Any ecological niche outside this window is called “extreme”, and organisms inhabiting them extremophiles
Figure 1.1

5. The environmental habitat (such as high salt or low pH) that a species inhabits is based on one main criterion
- The tolerance of that organism’s proteins and other macromolecular structures to the physical conditions within that niche
Note that multiple extremes in the environment can be met simultaneously
Figure 1.1 5

6. Global approaches used to study gene expression allow us to view how organisms respond to changes in their environment
- DNA microarrays assess which RNAs are made in a given organism at a given time or under a given condition
- Two-dimensional protein gels achieve separation of proteins based on differences in each protein’s isoelectric point (first dimension) and molecular weight (second dimension) 6

7. Figure 5.1 7

8. Table 5.1 8

9. Changes in Temperature
A bacterial cell’s temperature matches that of its immediate environment
Changes in temperature impact every aspect of microbial physiology
Each organism has an “optimum” temperature, as well as minimum and maximum temperatures that define its growth limits
Microbes that grow at higher temperatures can typically achieve higher rates of growth

10. Changes in Temperature
Microorganisms can be classified by their growth temperature
- Psychrophiles ~ 0-20o C
- Mesophiles ~ 15-45o C
- Thermophiles ~ 40-80o C
- Hyperthermophiles ~ o C
All of these organisms have membranes and proteins best suited for their temperatures 10

11. Figure 5.2 11

12. Figure 5.3
Figure 5.4 12

13. Heat-Shock Response
Rapid temperature changes experienced during growth activates batches of stress response genes
- Resulting in the heat-shock response
The protein products include chaperones that maintain protein shape and enzymes that change membrane lipid composition
This type of response has been documented in all living organisms examined so far 13

14. Variations in Pressure
Barophiles or piezophiles are organisms adapted to grow at very high pressures
- Up to 1,000 atm (101 MPa, or 14,000 psi)
Barotolerant organisms grow well over the range of 1-50 MPa, but their growth falls off thereafter
Note that many barophiles are also psychrophiles because the average temperature at the ocean floor is 2o C 14

15. Figure 5.5
Figure 5.6 15

16. Changes in Water Activity
Water activity (aw) is a measure of how much water is available for use
Osmolarity is a measure of the number of solute molecules in a solution, and is inversely related to aw
Aquaporins are membrane-channel proteins that allow water to traverse the membrane much faster than by diffusion
- Help protect the cell from osmotic stress 16

17. Minimizing Osmotic Stress
In addition to moving water, microbes have at least two mechanisms to minimize osmotic stress
- In hypertonic media, bacteria protect their internal water by synthesizing or importing compatible solutes (E.g.: Proline or K+)
- In hypotonic media, pressure-sensitive or mechanosensitive channels can be used to leak solutes out of the cell 17

18. Changes in Salt Concentrations
Halophiles require high salt concentrations
- From 2-4 M NaCl (10-20% NaCl)
- For comparison, seawater is ~ 3.5% NaCl
Figure 5.8 18

19. Changes in pH
Figure 5.11 19

20. Changes in pH
All enzyme activities exhibit optima, minima, and maxima with regard to pH
Bacteria regulate internal pH
- When environment is in a similar pH range
Weak acids can pass through membranes
- Disrupt cell pH homeostasis, and thus will kill cells
- This phenomenon is used to preserve foods 20

21. Changes in pH
Three classes of organisms are differentiated by the pH of their growth range
- Neutralophiles grow at pH 5-8
- Include most pathogens
- Acidophiles grow at pH 0-5
- Are often chemoautotrophs
- Alkaliphiles grow at pH 9-11
- Typically found in soda lakes 21

22. - It is also a major food for the famous pink flamingo
The cyanobacterium Spirulina has high concentrations of carotene, giving it a distinct pink color
- It is also a major food for the famous pink flamingo
Figure 5.15 22

23. pH Homeostasis
When cells are placed in pH conditions below the optimum, protons can enter the cell and lower internal pH to lethal levels
Microbes can prevent the unwanted influx of protons by exchanging extracellular K+ for intracellular H+ when the internal pH becomes too low
Under extremely alkaline conditions, the cells can use the Na+/H+ antiporter to bring protons into the cell in exchange for expelling Na+ 23

24. Figure 5.17 24

25. Oxygen As An Electron Acceptor
Many microorganisms use oxygen as a terminal electron acceptor in a process called aerobic respiration
Figure 5.18 25

26. Microbial Responses to Oxygen
Strict aerobes can only grow in oxygen
Microaerophiles grow only at lower O2 levels
Strict anaerobes die in least bit of oxygen
Facultative anaerobes can live with or without oxygen
Aerotolerant anaerobes grow in oxygen while retaining a fermentation-based metabolism

27. Oxygen-related growth zones in a standing test tube
Figure 5.19 27

28. Generation and destruction of reactive oxygen species (ROS)
Figure 5.20 28

29. Culturing Anaerobes in the Lab
Three oxygen-removing techniques are used today
1. Special reducing agents (thioglycolate) or enzyme systems (Oxyrase) can be added to ordinary liquid media
2. An anaerobe jar
3. An anaerobic chamber with glove ports
- O2 is removed by vacuum and replaced with N2 and CO2 29

30. Figure 5.21 30

31. Microbial Response to Starvation
Starvation is a stress that can elicit a “starvation response” in many microbes
- Enzymes are produced to increase the efficiency of nutrient gathering and to protect cell macromolecules from damage
This response is usually triggered by the accumulation of small signal molecules such as cAMP or guanosine tetraphosphate 31

32. Microbial Response to Starvation
Some organisms growing on nutrient-limited agar can even form colonies with intricate geometrical shapes that help the population cope, in some unknown way, to food stress
Figure 5.22 32

33. Oligotrophic Bacteria
In natural ecosystems, most microbes appear to be oligotrophs, organisms with a high rate of growth at low solute concentrations
- Indeed, they require low nutrient levels to survive
Some oligotrophic bacteria have thin extensions of their membrane and cell wall called prothecaes (stalks)
- These expand the surface area of the cell and increase nutrient-transport capacity 33

34. Humans Influence Microbial Ecosystems
Maximum diversity in an ecosystem is maintained, in part, by the different nutrient-gathering profiles of competing microbes
Figure 5.23 34

35. Humans Influence Microbial Ecosystems
Eutrophication is the sudden infusion of large quantities of a formerly limiting nutrient
- It can lead to a “bloom” of microbes, which can threaten the existence of competing species
Figure 5.24 35

36. Control of Microbes
A variety of terms are used to describe antimicrobial control measures
- Sterilization: Killing of all living organisms
- Disinfection: Killing or removal of pathogens from inanimate objects
- Antisepsis: Killing or removal of pathogens from the surface of living tissues
- Sanitation: Reducing the microbial population to safe levels 36

37. Microbes die at a logarithmic rate
Decimal reduction time (D value) is the length of time it takes an agent or condition to kill 90% of the population
Figure 5.25 37

38. Physical Agents High temperature
- Moist heat is more effective than dry heat
- Boiling water (100o C) kills most cells
- Killing spores and thermophiles usually requires a combination of high pressure and temperature
- Steam autoclave
- 121o C at 15 psi for 20 minutes 38

39. Figure 5.26 39

40. Physical Agents Pasteurization
- Many different time and temperature combinations can be used
- LTLT (low-temperature/long-time)
- 63o C for 30 minutes
- HTST (high-temperature/short-time)
- 72o C for 15 seconds
- Both processes kill Coxiella burnetii, the causative agent of Q fever 40

41. Physical Agents
Cold
- Low temperatures slow down growth and preserve strains
- Refrigeration temperatures (4-8o C) are used for food preservation
- For long-term storage of cultures
- Placing solutions in glycerol at -70o C
- Lyophilization or freeze-drying 41

42. Physical Agents Filtration
- Micropore filters with pore sizes of 0.2 mm can remove microbial cells, but not viruses, from solutions
Figure 5.27 42

43. Air can also be sterilized by filtration
Laminar flow biological safety cabinets force air through HEPA filters
Figure 5.28 43

44. Physical Agents Irradiation - Ultraviolet light
- Has poor penetrating power
- Used only for surface sterilization
- Gamma rays, electron beams and X-rays
- Have high penetrating power
- Used to irradiate foods and other heat- sensitive items 44

45. Chemical Agents
A number of factors influence the efficacy of a given chemical agent, including:
- The presence of organic matter
- The kinds of organisms present
- Corrosiveness
- Stability, odor, and surface tension 45

46. The Phenol Coefficient
The phenol coefficient test compares the effectiveness of disinfectants
Table 5.3 46

47. Commercial Disinfectants and Antiseptics
These include:
- Ethanol
- Iodine (Wescodyne and Betadine)
- Chlorine
- All of the above damage proteins, lipids, and DNA
- Are used to reduce or eliminate microbial content from objects 47

48. Figure 5.30 48

49. Antibiotics
Antibiotics are chemical compounds synthesized by one microbe that kill or inhibit the growth of other microbial species
Penicillin mimics part of the bacterial cell wall
- Prevents cell wall formation and is bactericidal
Figure 5.31 49

50. Effect of ampicillin (a penicillin derivative) on E. coli
Figure 5.32 50

51. Biological Control of Microbes
Biocontrol is the use of one microbe to control the growth of another
- Probiotics contain certain microbes that, when ingested, aim to restore balance to intestinal flora
- Lactobacillus and Bifidobacterium
- Phage therapy aims to treat infectious diseases with a virus targeted to the pathogen
- A possible alternative to antibiotics in the face of rising antibiotic resistance 51

52. Chapter Summary - Psychrophiles, mesophiles, and thermophiles
● Global analysis of genes and proteins allow us to study how microbes react to environmental changes
● Microbes are classified by growth temperature:
- Psychrophiles, mesophiles, and thermophiles
● Barophiles can grow at very high pressures
● Halophiles require high salt concentrations
● Microbes are classified by pH range:
- Acidophiles, neutralophiles, and alkaliphiles
● Microbes are classified by their O2 requirements:
- Aerobes, facultative, microaerophiles, and anaerobes 52

53. Chapter Summary
● Cells treated with antimicrobials die at a logarithmic rate
● Physical agents used to control microbes include:
- Autoclaving, Pasteurization, refrigeration, filtration, and irradiation
● Chemical agents used to control microbes include:
- Antiseptics and disinfectants
● Antibiotics selectively control microbial growth
● Biological control of microbes include the use of probiotics and phage therapy 53

54. Concept Quiz
Microbes that grow at temperatures between 40°C and 80°C are called:
a) psychrophiles.
b) mesophiles.
c) thermophiles.
d) extreme thermophiles.
Answer: C

55. Concept Quiz
Bacteria cannot grow in solutions with very high concentrations of sugar because
a) bacteria cannot digest pure sugar.
b) sugar raises the solution’s osmolarity.
c) sugar lowers the solution’s osmolarity
d) sugar raises the solution’s pH.
e) sugar lowers the solution’s pH.
Answer: B

56. Concept Quiz Physical agents used to prevent bacterial growth include:
a) pasteurization, freezing, phages.
b) irradiation, probiotics, filtration.
c) autoclaving, irradiation, freezing.
d) antibiotics, refrigeration, pasteurization.
Answer: C

57. Concept Quiz
The ______ coefficient test is used to compare disinfectants.
a) ethanol
b) iodine
c) phenol
d) chlorox
Answer: C

58. Concept Quiz Microbes that grow at very high pressures are called:
a) osmophiles.
b) mesophiles.
c) barophiles.
d) halophiles.
Answer: C

59. Concept Quiz
The D-value refers to the length of time it takes an agent to kill ___% of the microbial population.
a) 50
b) 90
c) 99
d) 100
Answer: C