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Microbial Physiology
Cultivation of microorganisms
Requirements for growth
Organic matter: C, H, O, N, P, S
Inorganic ions: K+, Na+, Fe2+,
Mg2+, Ca2+, and Cl-: required for
enzymatic catalysis and
maintaining chemical gradients
across the cell membrane.
Chemical energy
ATP
Ion motive force (eg. Proton
motive force)
Nutrition
Nutrients: synthetic vs. nonsynthetic media
Carbon source
Autotrophs (lithotrophs): use CO2 as the C source
Photosynthetic autotrophs: use light energy to reduce CO2.
Chemolithotrophs: use inorganics, such as H2 or thiosulfate
to reduce CO2.
Heterotrophs (organotrophs): use organic carbon (eg. glucose)
for growth.
Nitrogen source
Ammonium (NH4+) is used as the sole N source by most
microorganisms. For some bacteria, ammonium could be
produced from N2 by nitrogen fixation, or from reduction of
nitrate and nitrite.
Sulfur source
A component of several coenzymes and amino acids.
Most microorganisms can use sulfate (SO42-) as the S
source.
Phosphorus source
A component of ATP, nucleic acids, coenzymes, lipids,
teichoic acid, capsular polysaccharides; also is required
for signal transduction.
Phosphate (PO43-) is usually used as the P source.
Mineral source
Required for enzyme function.
For most microorganisms, it is necessary to provide
sources of K+, Mg2+, Ca2+, Fe2+, Na+ and Cl-. Many
other minerals (e.g., Mn2+, Mo2+, Co2+, Cu2+ and Zn2+)
can be provided in tap water or as contaminants of
other medium ingredients.
Acquisition of Fe is facilitated by production of
siderophores (hydroxamates and catechol derivatives).
Growth factors: organic compounds (e.g., amino
acids, sugars, nucleotides) a cell must contain in order
to grow but which it is unable to synthesize.
Fastidious microorganisms
Environmental factors
pH value
Neutrophils ( pH 6-8)
Acidophils ( pH 1-5)
Alkalophils ( pH 9-11)
Internal pH is regulated by
various proton transport systems
in the cytoplasmic membrane.
Temperature
Psychrophils ( 15-20 oC)
Mesophils ( 30-37 oC)
Thermophils ( at 50-60 oC)
Heat-shock response is induced
to stabilize the heat-sensitive
proteins of the cell.
Aeration
Obligate aerobes
Facultative anaerobes
Microaerophilics
Obligate anaerobes
(Capnophilics: bacteria that
do not produce enough CO2
and, therefore, require
additional CO2 for growth.)
Ionic strength and osmotic
pressure
Halophilic
Toxicity of O2 for anaerobes:
1. O2 reduced to H2O2 by enzymes.
2. O2 reduced to O2- by ferrous ion.
In aerobes and aerotolerant anaerobes, O2is removed by superoxide dismutase, while
H2O2 is removed by catalase.
Strict anaerobes lack both catalase and
superoxide dismutase.
Anaerobic cultivation methods
Excluding oxygen
Reducing agents
Anaerobic jar
Anaerobic glove chamber
Microbial metabolism
Intermediary metabolism
Assimilation (Anabolism)
Assimilatory pathways for the formation of key intermediates.
Biosynthetic sequences for the conversion of key
intermediates to end products.
Dissimilation (Catabolism)
Pathways that yield metabolic energy for growth and
maintenance.
Substrate-level
phosphorylation
Glycolysis
(EMP pathway)
Aerobic
respiration
Pyruvate: universal intermediate
Fermentation
Sources of metabolic energy
Substrate-level phosphorylation
Fermentation: metabolic process in
which the final electron acceptor is
an organic compound.
Respiration: chemical
reduction of an electron
acceptor through a specific
series of electron carriers in
the membrane. The electron
acceptor is commonly O2,
but CO2, SO42-, and NO3are employed by some
microorganisms.
Photosynthesis: similar to
respiration except that the
reductant and oxidant are
created by light energy.
Respiration can provide
photosynthetic organisms
with energy in the absence
of light.
Fermentation
In fermentation, the NADH
produced during glycolysis
is recycled to NAD.
Many bacteria are identified
on the basis of their
fermentative end products.
Fermentation of bacteria
produces yogurt, sauerkraut,
flavors to various cheeses
and wines.
Alcoholic fermentation is
uncommon in bacteria.
Saccharomycetes
Clostridium
Propionebacterium
E. coli
Enterobacter
Streptococcus
Lactobacillus
Function of TCA cycle
1. Generation of ATP
2. Supplies key intermediates for amino acids,
lipids, purines, and pyrimidines
3. The final pathway for the complete oxidation of
amino acids, fatty acids, and carbohydrates.
Pentose phosphate pathway
(hexose monophosphate shunt)
Functions:
1. Provides various
sugars as precursors
of biosynthesis, and
NADPH for use in
biosynthesis.
2. The various sugars
may be shunted back
to the glycolytic
pathway.
Cultivation methods
Medium
For microbiologic examination
Rich media
Use as many different media and conditions
of incubation as is practicable. Solid media
are preferred; avoid crowding of colonies.
Enrichment media
For isolation of a particular organism
Basic media
Selective media
Differential media
Agar: an acidic
polysaccharide
extracted from red
algae
Enrichment culture
Differential medium
Selective medium
Isolation of microorganisms in pure culture
Streak method
Pour plate method
For growing bacterial cells
Provide nutrients and conditions reproducing
the organism's natural environment.
Growth, survival and death of microorganisms
Most bacteria reproduce by
binary fission.
10-1
10-2
10-3
10-4
10-5
10-6
Measurement of microbial
concentrations:
Cell concentration (no. of
cells/unit vol. of culture)
0.1 ml
Viable cell count
Turbidimetric measurements
Biomass concentration (dry
wt. of cells/unit vol. of culture):
can be estimated by
measuring the amount of
protein or the volume
occupied by cells.
> 1000
220
18
Bacterial concentration:
220 x 106 x 10 = 2.2 x 109/ml
10-7
Bacterial growth curve
Lag phase (adaptation)
Exponential phase
Determination of the generation
time (doubling time)
The ending of this phase is due to
exhaustion of nutrients in the
medium and accumulation of toxic
metabolic products.
Stationary phase
A balance between slow loss of
cells through death and formation
of new cells through growth.
Alarmones is induced.
Death: irreversible loss of the
ability to reproduce.
Empirical test of death: culture of
Some bacteria undergo sporulation. cells on solid media.
Viable but nonculturable cells
Decline phase (the death phase)
Bacterial growth in nature
Interaction of mixed
communities
Biofilms
A natural environment may be
similar to a continuous culture.
Polysaccharide encased
community of bacteria attached
to a surface.
Bacteria grow in close
association with other kinds of
organisms.
Attachment of bacteria to a
surface or to each other is
mediated by glycocalyx.
The conditions in bacterial
close association are very
difficult to reproduce in the
laboratory. This is part of the
reason why so few
environmental bacteria have
been isolated in pure culture.
About 65% of human bacterial
infection involve biofilms.
Biofilms also causes problems
in industry.
Bioremediation is enhanced by
biofilms.
Biofilm: a community of microbes embedded in an organic polymeric
matrix (glycocalyx, DNA,, proteins), adhering to an inert or living surface.
Nucleic acid synthesis
1. Ribose-5-P (product of HMP)
synthesis of purine
ring from sugar moiety
inosine monophosphate
purine monophosphate
2. Pyrimidine orotate
orotidine monophosphate
(pyrimidine orotate attaches to ribose phosphate)
cytidine or urine (pyrimidine) monophosphate
3. Reduction of ribonucleotides at the 2’ carbon of the
sugar portion
deoxynucleotides