The Chemostat
Continuous culture
devices are a means of
maintaining cell
populations in
exponential growth for
long periods.
In a chemostat, the rate
at which the culture is
diluted governs the
growth rate and growth
yield.
1
Microbial Growth in a Chemostat
2
Microbial growth:
measurement and influence
of environmental factors
3
Measurement of Microbial Growth
Can measure changes in number of cells in a population
•
Direct cell counts
-counting chambers
-on membrane filters
•
Viable cell counts
-plating methods
-membrane filtration methods
Can measure changes in mass of population
-dry weight
-quantity of a particular cell constituent
-turbidometric measures
4
Counting chambers
• easy, inexpensive, and
quick
• cannot distinguish
living from dead cells
• examples: PetroffHauser or
hemocytometers
Figure 6.12
5
Direct counts on membrane
filters
• Cells filtered through special membrane
that provides dark background for
observing cells
• Cells are stained with fluorescent dyes
• Useful for counting bacteria
• With certain dyes, can distinguish living
from dead cells
6
Membrane filtration method
especially useful for analyzing aquatic samples
Figure 6.13
7
Measurement of Cell Mass
• Dry weight
– time consuming and not very sensitive
• Quantity of a particular cell constituent
– protein, DNA, ATP, or chlorophyll
• Turbidometric measures (light scattering)
– quick, easy, and sensitive
8
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more cells

more light
scattered

less light
detected
Figure 6.15
9
Environmental Factors on
Growth
• Most organisms grow in fairly
moderate environmental conditions
• Extremophiles
– grow under harsh conditions that
would kill most other organisms
10
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Table 6.3
11
Water Activity (aw) and
osmosis
• Water activity (aw)
– amount of water available to organisms
– reduced by interaction with solute
molecules (osmotic effect)
higher [solute]  lower aw
12
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Table 6.4
13
Halophilic and halotolerant
microorganisms
• Halophilic microorganisms
– Absolute requirement of salt for growth
– Accumulate K+ (primarily in archaea)
– Accumulate organic compounds (compatible
solutes) (primarily in bacteria)
• Halotolerant microorganisms
– No absolute requirement of salt for growth
– grow over wide ranges of salinity
– many use compatible solutes
14
• halophiles
– grow optimally at
> 0.2 M
• extreme halophiles
– require > 2 M
Figure 6.18
15
pH
• acidophiles
– growth optimum between pH 0 and pH 5.5
• neutrophiles
– growth optimum between pH 5.5 and pH 7
• alkalophiles
– growth optimum between pH 8.5 and pH 11.5
16
pH
• Most acidophiles and alkalophiles maintain an
internal pH near neutrality
– The plasma membrane is impermeable to protons
– Symport, antiport systems can be used to maintain
pH closer to neutrality
• Synthesize proteins that provide protection
– e.g., acid-shock proteins
• Many microorganisms change pH of their
habitat by producing acidic or basic waste
products
– most media contain buffers to prevent growth
inhibition
17
Temperature
• Greatly effects
enzyme activities.
• Organisms exhibit
distinct cardinal
growth temperatures
– minimal
– maximal
– optimal
Figure 6.20
18
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Figure 6.21
19
20
Hyperthermophiles
in Hot Springs
21
Adaptations of thermophiles
• Protein structure stabilized by a variety
of means
– more H bonds
– more proline
– chaperones
• Histone-like proteins stabilize DNA
• Membrane stabilized by variety of means
– more saturated, more branched and higher
molecular weight lipids, lipid monolayers
– e.g., ether linkages (archaeal membranes)
22
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Table 6.5
23
Oxygen Concentration
need
oxygen
Figure 6.22
24
prefer
oxygen
ignore
oxygen
oxygen is
toxic
< 2 – 10%
oxygen
Oxygen toxicity
• Some enzymes are extremely sensitive to
oxygen.
• oxygen easily reduced to toxic products
– superoxide radical
– hydrogen peroxide
– hydroxyl radical
• aerobes produce protective enzymes
– superoxide dismutase (SOD)
– catalase
25
26
27
Figure 6.24
28
Pressure
• barotolerant
– adversely affected by increased
pressure, but not as severely as
nontolerant organisms
• barophilic organisms
– require or grow more rapidly in the
presence of increased pressure
29
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Radiation
Figure 6.25
30
Radiation Damage
• Ionizing radiation
– X-rays and gamma rays
– mutations  death
– disrupts chemical structure of DNA
• damage may be repaired by DNA repair
mechanisms
31
Radiation Damage…
• Non-Ionization radiation
-Ultraviolet (UV) radiation
– mutations  death
– causes formation of thymine dimers in DNA
– DNA damage can be repaired by several
repair mechanisms
32
Radiation damage…
• Visible light
– at high intensities generates singlet
oxygen (1O2)
• powerful oxidizing agent
– carotenoid pigments protect many
light-exposed microorganisms from
photooxidation
33