Chapter4
Microbial growth
Environmental factors on growth
Growth in natural environment
Effect of environmental factors on growth
Water availability, pH, temperature, oxygen
concentration, pressure, radiation, and a
number of other environmental factors
influence microbial growth.
Yet many microorganisms, and particularly
bacteria, have managed to adapt and
flourish under environmental extremes that
would destroy most organisms.
Effect of temperature on bacterial growth rate
Bacteria grow over a
range of temperatures; they do
not reproduce below the
minimum growth temperature
nor above the maximum
growth temperature. Within
the temperature growth range
there is an optimum growth
temperature at which bacterial
reproduction is fastest.
Microorganisms are classified as psychrophiles,
mesophiles. thermophiles, and extremethemophiles
based on their optimal growth temperature.
Temperature effect:
1. Psychrophile
(0-20oC)
2. Mesophile
(45-15oC)
3. Thermophile
(40-70oC)
4. Hyperthermophile
(65-95oC)
5. Extrem hyperthermophile
(80-120oC)
Temperature
•
A most important factor influencing the growth
is the temperature sensitivity of enzyme
catalyzed reactions.
• High temperature:
1. Denature enzymes, transport carries, and other
proteins.
2. Microbial membranes are also disrupted by
temperature extremes.
3. The lipid bilayer melts and disintegrates.
Optimal growth temperature
• Mesophiles:
– human body temperature
* pathogens
* opportunists
• pyschrophile
– close to freezing
• thermophile
– close to boiling
Thermophile and Biotechnology
• Enzymes from thermophiles are capable of
catalyzing biochemical reactions at high
temperature, they are more stable;
• DNA polymerase, Taq polymerase, has been
isolated from thermophile-Thermus aquaticus, for
PCR application;
• DNA polymerase, pfu polymerase, isolated from
hyperthermophile-Pyrocuccus furiosus, is more
stable and useful for PCR application, it also less
prone to errors;
• Microbial process carried out at high T also
eliminate or greatly reduce cooling costs.
Molecular basis for growing at
extreme temperatures
• Molecular adaptations of enzymes to extreme
temperatures
– Cold-active enzymes are more flexible due to
higher portion of a helix and less amount of bsheet
– Heat resistant enzymes have different folding
patterns because of a few different amino acids
in some critical positions from their mesophile
counterpart, more salt bridges and more densely
packed hydrophobic interior
A-helix provides flexibility
B-sheet causes more rigidity
More b-sheets result in a more rigid
structure, which is less cold active
More a helixes result in more flexible
structure, which is cold resistant
Cell membrane must be in a fluidal
state in order to be functional
Hyperthermophiles have cell
membrane as lipid monolayer
Bacteria
Archaea
pH
• Many grow best at neutral pH
• Some can survive/grow: acid or alkali
• Commonly:
bacteria and protozoa: neutrophiles
algae and fungi: slight acidity.
actinomycetes: slight alkaline condition
pH effect on cell growth
• Organisms that live at low pH are called
acidophiles
• Fungi tend to be more acid-tolerant than
bacteria although their interior pH is close to
neutrality
• Several bacteria are obligate acidophilic (such
as Thiobacillus, Sulfolobus, Thermoplasma)
• A few organisms are alkalophilic (pH 10-11),
usually found in highly basic habitats such as
soda lakes and high carbonate soils.
• Microorganisms frequently change the pH
of their own habitat by producing acidic or
basic metabolic waste products.
• Buffers in media:
1. H2PO4-/HPO422. CaCO3
3. Peptides and amino acids
Solutes and water activity
• Compatible solutes: solutes that are compatible
with metabolism and growth when at high
intracellular concentrations.
• How do microorganisms adapt to hypotonic(低渗)
and hypertonic(高渗) environments?
1. The synthesis or uptake of choline(VB复合体),
betaine(甜菜碱), proline, glutamic acid, and other
amino acids.
2. Elevated levels of potassium ions.
3. Algae and fungi employ sucrose and polyols(多
羟复合物).
4. Protoza use contractile vacuoles.
Water activity (aw)
• Express quantitatively the degree of water
availability of the solution.
• P---vapor pressure
aw = P solution / P water
• aw is inversely related to osmotic pressure; if
a solution has high osmotic pressure, its aw
is low.
Water activity
The water activity of a solution is 1/100 the
relative humidity of the solution (when expressed as a
percent), or it is equivalent to the ratio of the solution's
vapor pressure to that of pure water.
Approximate lower aw limits for microbial growth:
0.90 – 1.00 for most bacteria, most algae and some fungi as
Basidiomycetes, Mucor, Rhizopus.
0.75 for Halobacterium, Aspergillus…
0.60 for some Saccharomyces species
Plasmolysis
If the concentration of solutes, such as sodium chloride,
is higher in the surrounding medium (hypertonic), then
water tends to leave the cell. The cell membrane shrinks
away from the cell wall (an action called plasmolysis), and
cell growth is inhibited.
Normal cell
Plasmolyzed cell
• osmotolerant: grow over wide ranges of water
activity or osmotic concentration.
• Microorganisms that have a specific requirement
for sodium ion are often called halophiles
• Organisms able to live in environment high in
sugar are called osmophiles
• Organisms capable of living in very salty
environments are called extreme halophiles
• Organisms able to live in very dry environments
are called xerophiles
Mechanism of Halophiles
1. Modified the structure of their proteins
and membranes rather that simply
increasing the intracellular concentrations
of solutes.
2. Accumulate enormous quantities of
potassium for stability and activity of the
enzymes, ribosomes and transport proteins.
Effects of Oxygen on Cell Growth
Terms used to describe O2 relationships of microorganisms:
• Aerobes:
– Obligate aerobes: oxygen presence necessary
– Facultative aerobes: oxygen not necessary, but
better with it
– Microaerophilic aerobes: required O2 at low level
than atmospheric
• Anaerobes:
– Aerotolerant: O2 not required, and growth no
better if O2 present
– Obligate (strict) anaerobes: Oxygen harmful or
lethal
1. Obligate aerobes
• grow in presence of oxygen;
• no fermentation;
• oxidative phosphorylation
2. Obligate anaerobes
•
•
•
•
no oxidative phosphorylation
fermentation
killed by oxygen
lack certain enzymes:
superoxide dismutase: O2-+2H+ to H2O2
catalase: H2O2 to H20 + O2
peroxidase: H2O2 to H20 /NAD to NADH)
3. Aerotolerant anaerobes
• respire anaerobically
• not killed by oxygen
4. Facultative anaerobes
• fermentation
• aerobic respiration
• survive in oxygen
5. Microaerophilic bacteria
• Grow low oxygen
• Killed high oxygen
Aerobic, anaerobic, facultative, microaerophilic and aerotolerant anaerobe growth
A small amount of agar has
been added to keep the
liquid from becoming
disturbed and the redox dye,
resazurin, which is pink
when oxidized and colorless
when reduced, is added as a
redox indicator.
The above results were
obtained after a reducing
agent such as thioglycolate
(巯基醋酸盐) was added to
the medium.
Resazurin (刃天青)，a redox
indicator dye, can be used to
detect oxygen in the
medium.
Different features
(a) Obligate aerobes-growth occurs only in the short
distance to which the oxygen diffuses into the medium.
(b) Facultative anaerobes growth is best near the surface,
where oxygen is available, but occurs throughout the tube.
(c) Obligate anaerobes-oxygen is toxic, and there is no
growth near the surface.
(d) Aerotolerant anaerobes-growth occurs evenly
throughout the tube but is not better at the surface because
the organisms do not use oxygen.
(e) Microaerophiles, aerobic organisms that do not tolerate
atmospheric concentrations of oxygen-growth occurs only
in a narrow band of optimal oxygen concentration.
How to culture
• Aerobic: culture vessel is shaken to aerate the medium
or sterile air must be pumped through the culture vessel.
• Anaerobes:
1. Media contain reducing agents such as
thioglycollate or crysteine . Boiling
2. Oxygen also may be eliminated from an anaerobic
system by removed air with a vacuum pump and
flushing out residual O2 with nitrogen gas.
3. GasPak jar: by using hydrogen and a palladium(钯)
catalyst to remove O2 through the formatin of water.
4. Plastic bags or pouchs: only a few samples pouch:
catalyst + calcium carbonateCO2
Toxic Forms of Oxygen
protein inactivation
• Singlet oxygen
• Superoxide anion (O2 )
• Hydrogen peroxide (H2O2)
.
• Hydroxyl radical (OH )
Enzymes that destroy toxic oxygen
(a) H2O2+H2O2
(b)
Catalase
过氧化氢酶
H2O2+NADH+H+
(c) O2-+O2-+2H+
2H2O+O2
Peroxidase
过氧化氢酶
2H2O+NAD+
Superoxide dismutase
过氧化氢歧化酶
H2O2 +O2
(d) 4O2-+4H+ Catalase+Superoxide dismutase 2H2O +3O2
How do you know a microbial cell contains catalase?
CATALASE TEST
Some bacteria and macrophages can reduce diatomic oxygen to
hydrogen peroxide or superoxide. Both of these molecules are toxic to
bacteria. Some bacteria, however, possess a defense mechanism which
can minimize the harm done by the two compounds. These resistant
bacteria use two enzymes to catalyze the conversion of hydrogen
peroxide and superoxide back into diatomic oxygen and water. One of
these enzymes is catalase and its presence can be detected by a simple
test. The catalase test involves adding hydrogen peroxide to a culture
sample or agar slant. If the bacteria in question produce catalase, they
will convert the hydrogen peroxide and oxygen gas will be evolved. The
evolution of gas causes bubbles to form and is indicative of a positive
test.
• Pressure (deep sea): nutrient recycling
barotolerant
barophilic
• Radiation:
sunlight:
visible light: photosynthetic
uv radiation: 260nm TT dimer
Infrared rays: heat
Radio waves:
ionizing: X-ray, gamma rays, spore mutation
Microbial Growth in natural
enviroments
• Viable but nonculturable vegetative
procaryotes
• Only 1-10% of observable cells are able to
form colonies.
• PCR
• Small subunit ribosomal RNA analysis
• Sensitive microscopic and isotopic
procedures
Quorum sensing and microbial
populations
• Quorum sensing: a phenomenon in which
bacteria monitor their own population density
through sensing the levels of signal molecules,
sometimes called autoinducers because they can
stimulate the cells that releases them.
• The bacteria reach a high population density
before they release enzymes. This is an advantage
within a host’s body as well as in the soil or an
aquatic habitat. eg. A pathogen
G- figure 6.19 P133
G+: an oligopeptide signal
• competence induction in Streptococcus
pneumoniae
• stimulation of sporulation by Bacillus subtilis.
Others:
• promote the formation of nature biofilms
• two different bacteria might stimulate each other
by releasing similar signals
Questions
•What environmental factors affect growth of
microorganisms?
•How to name microorganisms that grow best at a
temperature?
•How do you name microorganisms having
various O2 tolerance?
•What enzymes are involved in detoxifying toxic
active oxygen?
• Quorum sensing and microbial populations?