Cultivation of Microorganisms

KEY TERMS Obligate aerobe Obligate anaerobe Aerotolerant anaerobe Facultative anaerobe Microaerophilic Mesophile Thermophile Psychrophile Halophilic Osmophilic Medium

Bacterial Requirements for Growth Nutrients Oxygen (or absence) Energy Optimal temperature Optimal pH

SOURCES OF METABOLIC ENERGY The three major mechanisms for generating metabolic energy are Fermentation Respiration Photosynthesis At least one of these mechanisms must be employed if an organism is to grow

Fermentation The formation of ATP in fermentation is not coupled to the transfer of electrons Fermentation is a substrate phosphorylation, an enzymatic process in which a pyrophosphate bond is donated directly to ADP (adenosine diphosphate) by a phosphorylated metabolic intermediate The phosphorylated intermediates are formed by metabolic rearrangement of a fermentable substrate such as glucose, lactose, or arginine Example: fermentation of a molecule of glucose (C6H12O6) yields a net gain of two pyrophosphate bonds in ATP and produces two molecules of pyruvic acid (C3H6O3)

Fermentation Reaction

Respiration Respiration is chemical reduction of an oxidant (electron acceptor) through a specific series of electron carriers in the membrane establishes the proton motive force across the bacterial membrane Reductants (electron donor) may be organic or inorganic: For example, lactic acid serves as a reductant for some organisms, and hydrogen gas is a reductant for other organisms Oxidants Gaseous oxygen (O2) often is employed as an oxidant, but alternative oxidants that are employed by some organisms include carbon dioxide (CO2), sulfate (SO42–), and nitrate (NO3−)

Respiration in Eukaryotic Cell 36ATP

Respiration in Eukaryotic Cell

Photosynthesis Photosynthesis is similar to respiration in that the reduction of an oxidant via a specific series of electron carriers establishes the proton motive force Difference in the two processes in photosynthesis the reductant and oxidant are created photochemically by light energy absorbed by pigments in the membrane Photosynthesis can continue only as long as there is a source of light energy

Photosynthesis

Environmental Factors Affecting Growth Nutrients Hydrogen ion concentration (pH) Temperature Aeration Ionic strength & osmotic pressure

Nutrient Requirements Carbon Nitrogen Sulfur Phosphorus Metal ions (e.g. iron) Growth factors

Carbon Source Autotroph Heterotroph CO2 + Sunlight Photosynthesis CO2+H+SO42- Chemolithotroph Heterotroph Organic carbon eg., glucose Fermentation Respiration

Nitrogen Source The end products are NH4+ or NH3

Sulfur Source Most microorganisms can use sulfate as a sulfur source, reducing the sulfate to the level of hydrogen sulfide (H2S) Some microorganisms can assimilate H2S directly from the growth medium, but this compound can be toxic to many organisms

Phosphorus Source Phosphate (PO43−) is required as a component of ATP, nucleic acids, and such coenzymes as NAD, NADP, and flavins Many metabolites, lipids (phospholipids, lipid A), cell wall components (teichoic acid), some capsular polysaccharides, and some proteins are phosphorylated Phosphate is always assimilated as free inorganic phosphate (Pi)

Mineral Sources In formulating a medium for the cultivation of most microorganisms, it is necessary to provide sources of potassium, magnesium, calcium, and iron, usually as their ions (K+, Mg2+, Ca2+, and Fe2+) Many other minerals (eg, Mn2+, Mo2+, Co2+, Cu2+, and Zn2+) are required; these frequently can be provided in tap water or as contaminants of other medium ingredients

Growth Factors A growth factor is an organic compound which a cell must contain in order to grow but which it is unable to synthesize Some amino acids Some purines and pyrimidines Vitamins Different species of bacteria needs different growth factors

Nutrient Requirements Carbon Nitrogen Sulfur Phosphorus Metal ions (e.g. iron) Growth factors

Environmental Factors Affecting Growth Nutrients Hydrogen ion concentration (pH) Temperature Aeration Ionic strength & osmotic pressure

Hydrogen Ion Concentration (pH) Many grow best at neutral pH Some can survive/grow - Acid Alkali

Hydrogen Ion Concentration (pH) Most organisms (neutralophiles) grow best at a pH of 6.0–8.0, although some forms (acidophils) have optima as low as pH 3.0 and others (alkaliphiles) have optima as high as pH 10.5 Microorganisms regulate their internal pH over a wide range of external pH values Acidophiles maintain an internal pH of about 6.5 over an external range of 1.0–5.0 Neutralophiles maintain an internal pH of about 7.5 over an external range of 5.5–8.5 Alkaliphiles maintain an internal pH of about 9.5 over an external range of 9.0–11.0

Temperature Psychrophilic forms grow best at low temperatures (15–20oC) Mesophilic forms grow best at 30–37oC Most organisms are mesophilic 30oC is optimal for many free-living forms, and the body temperature of the host is optimal for symbionts of warm-blooded animals Thermophilic forms grow best at 50–60oC

Optimal Growth Temperature Mesophile human body temperature pathogens opportunists Pyschrophile close to freezing Thermophile close to boiling

Heat Shock / Cold Shock Microorganisms share with plants and animals the heat-shock response, a transient synthesis of a set of “heat-shock proteins,” (HSP) when exposed to a sudden rise in temperature above the growth optimum HSPs appear to be unusually heat-resistant and to stabilize the heat-sensitive proteins of the cell Bacteria also exhibit a phenomenon called cold shock: the killing of cells by rapid cooling For example, the rapid cooling of Escherichia coli from 37 oC to 5oC can kill 90% of the cells A number of compounds protect cells from either freezing or cold shock: glycerol, dimethyl sulfoxide (DMSO)

Aeration Obligate aerobe Facultative anaerobe Obligate anaerobe specifically requiring oxygen as hydrogen acceptor Facultative anaerobe able to live aerobically or anaerobically Obligate anaerobe requiring a substance other than oxygen as hydrogen acceptor and being sensitive to oxygen inhibition

Obligate Aerobes No fermentation Oxidative phosphorylation Grow in presence of oxygen No fermentation Oxidative phosphorylation

Obligate Anaerobes Fermentation No oxidative phosphorylation Killed by oxygen The natural by-products of aerobic metabolism are the reactive compounds hydrogen peroxide (H2O2) and superoxide (O2-) Lack certain enzymes superoxide dismutase O2-+2H+ H2O2 catalase H2O2 H20 + O2 peroxidase H2O2 H2O /NAD NADH

Facultative Anaerobes Fermentation Aerobic respiration Survive in oxygen

Microaerophilic Bacteria Grow low oxygen Killed high oxygen

Ionic Strength and Osmotic Pressure For most organisms, the properties of ordinary media are satisfactory; however, for marine forms and organisms adapted to growth in strong sugar solutions, these factors must be considered Halophilic Organism: requires high salt concentrations Osmophilic Organism: requires high osmotic pressures

Cultivation Methods Two problems will be considered Media: the choice of a suitable medium Cultivation Methods: the isolation of a bacterial organism in pure culture

Cultivation Medium: Based on Purpose Basic medium Enrichment medium Selective medium Differentiation medium Anaerobic medium

Cultivation Medium: Based on physical properties Liquid medium Solid medium Semi-Solid medium

Medium Applications Liquid Medium Pure Culture Amplifying culture Semi-solid medium Motion: flagellum Solid Medium Pure culture Isolation culture colony

Colony Attributes