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.

Microbial Growth in a Chemostat

Microbial growth: measurement and influence of environmental factors

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

Counting chambers easy, inexpensive, and quick cannot distinguish living from dead cells examples: Petroff-Hauser or hemocytometers Figure 6.12

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

Membrane filtration method especially useful for analyzing aquatic samples Figure 6.13

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

more cells  more light scattered less light detected Figure 6.15 Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. more cells  more light scattered less light detected Figure 6.15

Environmental Factors on Growth Most organisms grow in fairly moderate environmental conditions Extremophiles grow under harsh conditions that would kill most other organisms

Copyright © McGraw-Hill Companies, Inc Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 6.3

Water Activity (aw) and osmosis amount of water available to organisms reduced by interaction with solute molecules (osmotic effect) higher [solute]  lower aw

Copyright © McGraw-Hill Companies, Inc Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 6.4

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

halophiles extreme halophiles grow optimally at > 0.2 M require > 2 M Figure 6.18

pH acidophiles neutrophiles alkalophiles 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

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

Temperature Figure 6.20 Greatly effects enzyme activities. Organisms exhibit distinct cardinal growth temperatures minimal maximal optimal Figure 6.20

Copyright © McGraw-Hill Companies, Inc Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 6.21

Hyperthermophiles in Hot Springs

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)

Copyright © McGraw-Hill Companies, Inc Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Table 6.5

Oxygen Concentration need oxygen ignore oxygen < 2 – 10% oxygen prefer oxygen oxygen is toxic Figure 6.22

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

Figure 6.24

Pressure barotolerant barophilic organisms 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

Copyright © McGraw-Hill Companies, Inc Copyright © McGraw-Hill Companies, Inc. Permission required for reproduction or display. Radiation Figure 6.25

Radiation Damage Ionizing radiation X-rays and gamma rays mutations  death disrupts chemical structure of DNA damage may be repaired by DNA repair mechanisms

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

Radiation damage… Visible light at high intensities generates singlet oxygen (1O2) powerful oxidizing agent carotenoid pigments protect many light-exposed microorganisms from photooxidation