Cultivation of microorganisms Requirements for growth Organic matter: C, H, O, N, P, S Inorganic ions: K +, Na +, Fe 2+, Mg 2+, Ca 2+, 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 CO 2 as the C source Photosynthetic autotrophs: use light energy to reduce CO 2. Chemolithotrophs: use inorganics, such as H 2 or thiosulfate to reduce CO 2. Heterotrophs (organotrophs): use organic carbon (eg. glucose) for growth. Nitrogen source Ammonium (NH 4 + ) is used as the sole N source by most microorganisms. Ammonium could be produced from N 2 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 (SO 4 2- ) 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 (PO 4 3- ) is usually used as the P source.

Mineral source Required for enzyme function. For most microorganisms, it is necessary to provide sources of K +, Mg 2+, Ca 2+, Fe 2+, Na + and Cl -. Many other minerals (e.g., Mn 2+, Mo 2+, Co 2+, Cu 2+ and Zn 2+ ) 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 various proton transport systems in the cytoplasmic membrane. Temperature Psychrophils ( o C) Mesophils ( o C) Thermophils ( at o C) 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 CO 2 and, therefore, require additional CO 2 for growth.) Ionic strength and osmotic pressure Halophilic

Toxicity of O 2 for anaerobes: 1. O 2 reduced to H 2 O 2 by enzymes. 2. O 2 reduced to O 2 - by ferrous ion. In aerobes and aerotolerant anaerobes, O 2 - is removed by superoxide dismutase, while H 2 O 2 is removed by catalase. Strict anaerobes lack both catalase and superoxide 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 reduction of an electron acceptor through a specific series of electron carriers in the membrane. The electron acceptor is commonly O 2, but CO 2, SO 4 2-, and NO 3- 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 PropionebacteriumEnterobacter 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.

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. Pentose phosphate pathway (hexose monophosphate shunt)

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 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 culture Differential medium Selective medium Isolation of microorganisms in pure culture Pour plate method Streak 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 > Bacterial concentration: 220 x 10 6 x 10 = 2.2 x 10 9 /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.