Cultivation of microorganisms Requirements for growth Organic matter: C, H, O, N, P, S Inorganic...
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Transcript of Cultivation of microorganisms Requirements for growth Organic matter: C, H, O, N, P, S Inorganic...
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
Proton motive force
Microbial Physiology
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 micro
organisms. 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 s
ource.
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 so
urces 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.
Uptake 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
pH value Neutrophils ( pH 6-8) Acidophils ( pH 1-5) Alkalophils ( pH 9-11) Internal pH is regulated by variou
s 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.
Environmental factors
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, O2- i
s removed by superoxide dismutase, while H
2O2 is removed by catalase.
Strict anaerobes lack both catalase and sup
eroxide dismutase.
Excluding oxygen
Reducing agents
Anaerobic jar
Anaerobic glove chamber
Anaerobic cultivation methods
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.
Pyruvate: universal intermediate
Aerobic respiration
Fermentation
Glycolysis (EMP pathway)
Substrate-level phosphorylation
Fermentation: metabolic process in which the final electron acceptor is an organic compound.
Sources of metabolic energy Respiration: chemical reduct
ion of an electron acceptor through a specific series of electron carriers in the membrane. The electron acceptor is commonly O2, but CO2, SO4
2-, and NO3- are 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.
Substrate-level phosphorylation
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.
Fermentation
Saccharomycetes
E. coliClostridium
Propionebacterium Enterobacter
StreptococcusLactobacillus
Function of TCA cycle
1. Generation of ATP
2. Supplies key intermediates for amino acids, li
pids, purines, and pyrimidines
3. The final pathway for the complete oxidation of
amino acids, fatty acids, and carbohydrates.
Functions:
1. Provides various suga
rs as precursors of bio
synthesis, and NADP
H for use in biosynthe
sis
2. The various sugars m
ay be shunted back to
the glycolytic pathway.
Pentose phosphate pathway (hexose monophosphate shunt)
1. Ribose-5-P (product of HMP) synthesis of purine r
ing from sugar moiety inosine monophosphate
purine monophosphate
2. Pyrimidine orotate orotidine monophosphate (pyri
midine orotate attaches to ribose phosphate)
cytidine or urine (pyrimidine) monophosphate
3. Reduction of ribonucleotides at the 2’ carbon of the sug
ar portion deoxynucleotides
Nucleic acid synthesis
Cultivation methods
Medium
Basic media
Rich media
Enrichment media
Selective media
Differential media
Agar: an acidic polysaccharide extracted from red algae
For microbiologic examination
Use as many different media and conditions of incubation as is practicable. Solid media are preferred; avoid crowding of colonies.
For isolation of a particular organism
Enrichment cultureDifferential mediumSelective medium
Isolation of microorganisms in pure culture
Pour plate methodStreak method
For growing bacterial cells
Provide nutrients and conditions reproducing the organism's natural environment.
Most bacteria reproduce by binary fission.
Measurement of microbial concentrations:
Cell concentration (no. of cells/unit vol. of culture)
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.
Growth, survival and death of microorganisms
0.1 ml
10-1 10-2 10-3 10-4 10-5 10-6 10-7
> 1000 220 18
Bacterial concentration:
220 x 106 x 10 = 2.2 x 109/ml
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.
Some bacteria undergo sporulation.
Decline phase (the death phase)
Bacterial growth curve
Death: irreversible loss of the ability to reproduce.
Empirical test of death: culture of cells on solid media. Viable but nonculturable cells
Bacterial growth in nature
Interaction of mixed communities
A natural environment may be similar to a continuous culture.
Bacteria grow in close association with other kinds of organisms.
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.
Biofilms
Polysaccharide encased community of bacteria attached to a surface.
Attachment of bacteria to a surface or to each other is mediated by glycocalyx.
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, slime), adhering to an inert or living surface.