1

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.

2

Microbial Growth in a Chemostat

3

Microbial growth: measurement and influence

of environmental factors

4

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

5

Counting chambers

• easy, inexpensive, and quick

• cannot distinguish living from dead cells

• examples: Petroff-Hauser or hemocytometers

Figure 6.12

6

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

7

Membrane filtration method

especially useful for analyzing aquatic samples

Figure 6.13

8

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

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9

more cells

more lightscattered

less lightdetected

Figure 6.15

10

Environmental Factors on Growth

• Most organisms grow in fairly moderate environmental conditions

• Extremophiles– grow under harsh conditions that

would kill most other organisms

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11

Table 6.3

12

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

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13

Table 6.4

14

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

15

• halophiles– grow optimally at

> 0.2 M

• extreme halophiles– require > 2 M

Figure 6.18

16

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

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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

18

Temperature

• Greatly effects enzyme activities.

• Organisms exhibit distinct cardinal growth temperatures

– minimal

– maximal

– optimal

Figure 6.20

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19

Figure 6.21

20

21

Hyperthermophilesin Hot Springs

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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)

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23

Table 6.5

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Oxygen Concentration

needoxygen

preferoxygen

ignoreoxygen

oxygen istoxic

< 2 – 10%oxygen

Figure 6.22

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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

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27

28

Figure 6.24

29

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

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30

Radiation

Figure 6.25

31

Radiation Damage

• Ionizing radiation– X-rays and gamma rays

– mutations death

– disrupts chemical structure of DNA• damage may be repaired by DNA repair

mechanisms

32

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

33

Radiation damage…

• Visible light– at high intensities generates singlet

oxygen (1O2)

• powerful oxidizing agent

– carotenoid pigments protect many light-exposed microorganisms from photooxidation