The Continuous Culture of
Microorganisms:
n
A microbial population of can be maintained in the
exponential growth phase and at a constant biomass concentration for extended
periods.
n
Open System: system with constant environmental conditions maintained through continual provision of
nutrients and removal of wastes .
Two
Common Major Types of Continuous Culture Systems:
(1). Chemostats
Ø
Sterile medium is fed into the culture vessel at the
same rate as the media containing the Mos is removed.
(2). Turbidostats
Ø Photocell that measures the absorbance or
turbidity of the culture in the growth vessel.
Environmental Factors on
Growth:
The growth of Mos
are effected by Chemical and Physical surroundings:
Live
1.5 miles below the earth’s surface, w/o oxygen, and below 60° C.
Temperature
Ø
Microbial cell
temperature directly reflects that of the cell’s surrounding.
§
Most bacteria can
grow over a temperature range of
about 30˚ or more but have a
narrow range for optimal growth.
§
As we decrease
the temperature below the optimum, we see a decline in growth rate that is
consistent with enzymatic activity, but
then it becomes very steep, giving rise to a fairly well defined minimal growth
temperature.
§
Above the optimum
temperature, we see the growth rate decline very steeply, which gives rise to a
sharply defined maximum growth temperature
It is not known what sets the upper and lower temperature although they are thought to
§
reflect
properties of the membrane lipids,
§
effects on
protein conformation, and/or initiation of protein synthesis.
Temperature
Sensitivity of Enzyme-Catalyzed Reactions:
§
A temp rise,
increases the growth rate due to the velocity of an enzyme-catalyzed reaction.
§
Velocity will
double for every 10º C rise in temperature.
§
As rate increase,
the metabolism is more active at higher temp, Mo grow faster.
§
Example: 10
-- 30, Velocity is 15 What is
the velocity of the cell at 50º C?
High
Temperatures:
§ Damage
MOs by denaturing enzymes, transport carriers, and other proteins
§ Membranes are disrupted, lipid bilayer simply melts and disintergrates.
Low
Temperatures:
§
membranes
solidify and enzymes don’t work properly.
The temperature range of an organism can be used as a
classifying characteristic. All
bacteria have distinct cardinal temperatures:
Cardinal Temperatures Growth Temperatures:
§
Minimum
§
Optimum
§
Maximum
Pc can grow at much higher Temp than EC
Psychrophiles can grow at temperatures between 0-20º,
optima growth is 15º
§
Frequently found
in naturally cold waters and soils.
Such
as the Artic and Antarctic.
§
Examples include
the Pseudomonads and Bacillus,
§ Enzymes, transport systems and protein
synthetic mechanisms function well as low temp.
§ Cell membrane have high levels of
unsaturated fatty acid and remain semifluid when cold.
Psychrotrophs
or Facultative Psychrophiles can
grow at 0 to 7 º C.
§ Optima
20-30 º C
§ Maxima 35 º C
§ Psychrotrophic bacteria and fungi are important in spoilage of refrigerated foods
Most bacteria are Mesophiles and grow between 20-45 ºC.
§
Those that are
found in the mammalian body have an optimum temperature of 37-44 º C, Maxima is 45 º C.
§ Those found in the environment have an optimum
of about 30 degrees C
§ Almost all human pathogens are mesophiles,
env. is around 37ºC.
Thermophiles grow at
temperature of 55º C or higher.
Minimum of 45 ºC and
optima between 55 and 65 º C.
§
Majority of
prokaryotes
§
Flourish in
composts, self-heating hay stacks, hot water lines, and hot springs.
§
More heat stable
enzymes and protein synthesis systems / funct at higher temp.
§
Membranes lipids
more saturated and have higher melting points causing membrane to remain in
tact at higher temp.
§
These organisms
are extremely useful in that they serve as sources for exceptionally stable
forms of enzymes (i.e. bacillus stearothermophilus)
Hyperthermophiles are
thermophiles that can grow at 90º C or above,
§
Procayotes gowth
optima between 80 and 113 º C.
§
Do not grow well
below 55 º C.
pH (Acidity or alkalinity)
pH is a
measure of the Hydrogen Ion activity of a solution and is defined as the
negative logarithm of the hydrogen ion concentration (expressed in terms of
molarity).
pH scale
0.0 (1.0 M H+) – 14.0 (1.0 x
10 -14M H+)
Each unit
represents a tenfold change in hydrogen ion concentration.
§ Bacteria can also be classified by the pH ranges in which they grow.
§ The internal pH of the cell remains close to neutral,
§ An organism’s tolerance to fluctuations in pH reflects the capacity of the membrane
pumps to maintain that pH
Acidophiles grow best below pH 4.0.
§ The vinegar forming acetobacter and some of the sulfur oxidizing bacteria can
tolerate the pH values as low as ~0 (the pH of 1N sulfuric acid)
§ Growth range between 0 – 5.5
Neutrophiles
§ Growth range is 5.5 to 8.0
§
Most bacteria
and protozoa
Alkalophiles (most grow best above ~pH 10)
§ Growth range 8.5 to 11.5
§ a few of the urea splitters, Alcaligenes faecalis, and vibrio cholerae can thrive at pH levels as high as 9 and can tolerate levels greater than 10.
§ E. coli cannot withstand pH conditions greater than 8 or below 4.5.
a. H2O « H+ + OH-
[H+] = [OH-] = 1.00 x 10-7
mole per liter in pure water.
b. pH =
log 1/[H+]
The
pH of pure water is log (1)/(1.00 x 10-7)
= log (1.00 x 107) = 7.00.
c. Acids are
pH 0 - 7, bases are pH 7 - 14.
d. Every increase
of 1 pH unit is a 10-fold decrease in [H+].
Most bacteria grow between pH 5 and pH
8 (across a 1,000-fold difference in external hydrogen ion concentration).
A few extreme
acidophiles (oddly, all are eukaryotes and archaea) can grow at or near pH
0.
Oxygen Concentration
Oxygen effects on growth
a. O2 reacts with certain enzymes in the cell to form hydrogen peroxide
(H2O2) and superoxide (O2-). These compounds can damage biological macromolecules.
b. Detoxifying enzymes: catalase,
peroxidase, and superoxide dismutase.
c. Singlet oxygen (esp. produced in
photosynthesis) can be quenched by carotenoids
d. Categories of microorganisms (by oxygen environment):
(1) Strict aerobes:
§
Organism
grow in the presence of atmospheric O2
(2) Strict anaerobes
§
Do
not tolerate O2 at all and die in the presence of it.
(3) Facultative anaerobes
§
Do
not require O2 for growth but do grow better in its presence.
(4) Aerotolerant anaerobes
§
Grow
equally well whether it is present or not
(5) Microaerophiles (low oxygen)
§
damaged
by the normal atmospheric level of O2 (20%) and require O2 levels below the range of 2 to 10% for growth.
Most organism
are very sensitive to the salinity of the environment or osmotic pressure.
The rigid structure
of the bacterial cell wall enables it to grow over a wide range of osmotic
pressures.
Most bacteria
grow in ranges between 0.85% NaCl (physiological saline) and 3.5% NaCl
(seawater)
Some bacteria
have adapted mechanisms which enable them to withstand extremes of osmotic
pressure
Halophiles
grow best in environments where the osmotic pressure ranges between 18-24%
NaCl.
Salinity (osmotic balance):
§
[hi
H2O in/ low H2O out (hi salt) =>becomes dehydrated;
§
low
salt environment, H2O rushes in and cell lyses
§
Many
microbial environments are salty. For
example, the oceans cover about 71% of the Earth’s surface. Cells must maintain an osmotic balance with
the environment while not allowing high salt concentrations to inhibit
essential metabolic processes.
§
Salt
concentration in human blood is about 0.85% NaCl (physiological saline).
§
Sea
water contains about 3.5% salt (mostly NaCl).
The Dead Sea has a salt concentration of about 24%.
§
A
saturated salt solution contains about 35% NaCl.
§
Microorganisms
that grow in extreme high-salt environments (156-30% NaCl; salt pans,
evaporating pools, etc.) are called halophiles .
halophile vs. halotolerant
d. Compatible solutes are produced to increase solute levels
within cell without inhibiting cellular processes allowing the
e. Bacteria in the genus Halobacterium
grow best in environments where the NaCl concentration is above 18%. They actively pump Na+ ions out
of the cell and pump K+ ions in to maintain their osmotic
balance. Energy for pumping is
generated by a unique light-dependent photosynthetic process that is not based
on chlorophyll.
Growth of bacterial cell cultures (populations)
1. Doubling time or generation time: the time between one cell division and the
next (the time it takes for the population of cells to double in size).
a. Doubling time depends upon the species and the culture
conditions.
b. Optimal doubling times:
E. coli (20 minutes) versus Mycobacterium
tuberculosis (15-16 hours).
c. Natural environments:
E. coli in the human intestine (10 hours), Pseudomonas aeruginosa in
soil (2-3 days).
How does a bacterial population grow?
a. c. Conclusion: the number of cells will double with each
new generation (exponential growth).
d. Exponential growth equation
N2 = N1 2n equation
(1)
N2 = the number of bacterial cells at time t2
N1 = the number of bacterial cells at time t1
n = the number of doublings
e. The logarithmic form of the equation:
log N2 = log N1 + n log 2 equation (2)
f. Definition of doubling time:
g = (t2 - t1)/n equation (3)
g. Substitute equation (3) into equation (2):
log N2 = log N1 + log 2 (t2 - t1)/g equation (4)
h. Substitute log 2 = 0.30:
log N2 = log N1 + 0.30 (t2 - t1)/g equation (5)
i. Usually N2, N1, and (t2 - t1) are measured
experimentally and we solve equation (5) for g. However, we can solve the equation for any unknown if all
of the other quantities are given.
j. Example: for a culture
of E. coli: let g = 20 minutes, N1 = 1 cell, and (t2 - t1) = 2 days (2,880
minutes). What is N2?
(log 1 = 0)
log N2 = (log 1)+0.30 (2,880 minutes)/(20 minutes)= 43.2
N2 = 1.6 x 1043 cells
If each cell weighs 1 x 10-12 grams:
(1.6 x 1043 cells) (1 x 10-12 g/cell) = 1.6 x 1031 g
= approximately 2,700 times
the total mass of the Earth (6 x 1027 grams)