Chapter 5
Environmental Influences and Control of
Microbial Growth
1
Chapter Overview
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
●
2
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.
3
Environmental Influences and Control of
Microbial Growth
Lactobacillus plantarum
pH 6.5
Lactobacillus plantarum
pH 3
4
Environmental Limits of
Microbial Growth
“Normal” growth conditions
- Sea-level; temperature 20–40oC; neutral
pH; 0.9% salt; and ample nutrients
Any ecological niche outside this window is
called “extreme,” and organisms
inhabiting them are called extremophiles.
Figure 1.1
5
Genomic and Proteomic approaches to
study gene expressions
Global approaches used to study gene expression
allow us to view how organisms respond to
changes in their environment.
- DNA microarrays assess which RNAs
(Transcripts) are made in a given organism
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).
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Response to environmental stress: Global analysis: Genes and proteins expressed
under two different growth conditions.
The microarrays contain all genes on a genome attached to a slide. Organisms
grown under two growth conditions. Transcripts (RNAs) are converted to cDNA and
hybridized to the slide. Red indicate gene expressed under one conditions and
Green under different conditions. Yellow shows expression under both conditions.
The 2-D gel image shows protein expressed under 2 pH conditions
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8
Effect of Temperature
•
•
Changes in temperature impact every aspect of
microbial physiology.
Each organism has an “optimum, minimum and
maximum” temperatures that define its growth
limits. These are cardinal temperatures
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Temperature Optimum
Microorganisms can be classified by their
growth temperature:
- Psychrophiles ~ 0–20oC (15oC)
- Mesophiles ~ 15–45oC (37 oC)
- Thermophiles ~ 40–80oC (60 oC)
- Hyperthermophiles ~ 65–121oC (90oC)
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11
Psychrophiles can be found in
Icebergs
Yellowstone hotspring
Bacterium from South Polar
snow
Thermus aquatics,
Yellowstone
Methanocaldococcus jannaschii,
780 C and 30 psi. Isolated from
2600 m deep “white smoker” 12
chimney
How do microbes deal with cold?
• Cytoplasmic membrane contain high-level of
•
•
unsaturated fatty acids allowing their
membranes to be more flexible in cold.
Contain antifreeze proteins and
cryoprotectants such as trehalose
Enzymes from psychrophiles are useful for
biotechnology such a bioremediation and
for biochemical reactions at low temperature
Trehalose
13
Cytoplasmic membrane contain high-level of unsaturated
fatty acids allowing their membranes to be more flexible in
cold.
14
How do microbes deal with heatShock?
• Rapid temperature changes experienced by
•
•
•
microbes show the expression of certain genes.
The proteins, chaperones help maintain protein
shape (3-D structure).
Specialized DNA binding proteins protect DNA
from denaturation.
Membranes have high levels of saturated fatty
acids or hydrocarbons. Some archaea have
single lipid layer in membrane as opposed to
bilayer
15
Archaeal Lipids
Extreme temperature
archaea
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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 2oC.
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Figure 5.5
Figure 5.6
18
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
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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.
20
Water Activity (aw)
aw = Vapor Pressure of a Solution
Vapor Pressure of Pure water
aw of solutions ranges from 0-1.
aw of pure water is 1 (100% humidity).
21
Changes in Salt Concentrations
Halophiles require high salt concentrations.
- From 2–4 M NaCl (10–20% NaCl)
- For comparison, seawater is ~ 3.5% NaCl
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Changes in pH
Figure 5.11
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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.
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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
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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
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pH Homeostasis
When cells are placed in low pH conditions,
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+.
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Figure 5.17
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Microbial Responses to Oxygen
Strict aerobes can only grow in oxygen.
Microaerophiles grow only at lower O2 levels.
Strict anaerobes die in the least bit of oxygen.
Facultative anaerobes can live with or without
oxygen.
Aerotolerant anaerobes grow in oxygen while
retaining a fermentation-based metabolism.
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Oxygen-related growth zones in a standing
test tube
Figure 5.19
30
Generation and destruction of reactive
oxygen species (ROS)
Figure 5.20
31
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.
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Figure 5.21
33
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.
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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
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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 prothecae (stalks).
- These expand the surface area of the cell
and increase nutrient-transport capacity.
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Humans Influence Microbial Ecosystems
Maximum diversity
in an ecosystem
is maintained, in
part, by the
different nutrientgathering profiles
of competing
microbes.
Figure 5.23
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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
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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
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Microbes die at a logarithmic rate.
Decimal reduction time (D value) is the
length of time it takes an agent or a
condition to kill 90% of the population.
Figure 5.25
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Physical Agents
High temperature
- Moist heat is more effective than dry heat.
- Boiling water (100oC) kills most cells.
- Killing spores and thermophiles usually
requires a combination of high pressure and
temperature.
- Steam autoclave
- 121oC at 15 psi for 20 minutes
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Figure 5.26
42
Physical Agents
Pasteurization
- Many different time and temperature
combinations can be used.
- LTLT (low-temperature/long-time)
- 63oC for 30 minutes
- HTST (high-temperature/short-time)
- 72oC for 15 seconds
- Both processes kill Coxiella burnetii, the
causative agent of Q fever.
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Physical Agents
Cold
- Low temperatures slow down growth and
preserve strains.
- Refrigeration temperatures (4–8oC) are
used for food preservation.
- For long-term storage of cultures
- Placing solutions in glycerol at –70oC
- Lyophilization or freeze-drying
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Physical Agents
Filtration
- Micropore filters with pore sizes of 0.2 mm
can remove microbial cells, but not viruses,
from solutions.
Figure 5.27
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Air can also be sterilized by filtration.
Laminar flow biological safety cabinets
force air through HEPA filters.
Figure 5.28
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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 heatsensitive items
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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
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The Phenol Coefficient
The phenol coefficient test compares the
effectiveness of disinfectants.
Table 5.3
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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
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Figure 5.30
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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
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Effect of ampicillin (a penicillin derivative) on
E. coli
Figure 5.32
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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
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Chapter Summary
Global analysis of genes and proteins allows 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
●
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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 includes the use of
probiotics and phage therapy.
●
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