Microbial Nutrition & Growth A.Nutrient Requirements B.Nutrient Transport Processes C.Culture Media Growth in Batch Culture Mean Generation Time and Growth Rate Measurement of Microbial Growth Continuous Culture Factors Influencing Growth Nutrient Requirements Energy Source Phototroph
Uses light as an energy source Chemotroph Uses energy from the oxidation of reduced chemical compounds Nutrient Requirements Electron (Reduction potential) Source
Organotroph Uses reduced organic compounds as a source for reduction potential Lithotroph Uses reduced inorganic compounds as a source for reduction potential Nutrient Requirements Carbon source
Autotroph Can use CO2 as a sole carbon source (Carbon fixation) Heterotroph Requires an organic carbon source; cannot use CO2 as a carbon source Nutrient Requirements
Nitrogen source Organic nitrogen Oxidized forms of inorganic nitrogen Nitrate (NO32-) and nitrite (NO2-) Reduced inorganic nitrogen
Primarily from the catabolism of amino acids Ammonium (NH4+) Dissolved nitrogen gas (N2) (Nitrogen fixation) Nutrient Requirements Phosphate source Organic phosphate Inorganic phosphate (H2PO4- and HPO42-)
Sulfate (SO42-) Sulfide (S2- or H2S) Elemental sulfur (So) Nutrient Requirements Special requirements Amino acids Nucleotide bases Enzymatic cofactors or vitamins
Nutrient Requirements Prototrophs vs. Auxotrophs Prototroph A species or genetic strain of microbe capable of growing on a minimal medium consisting a simple carbohydrate or CO2 carbon source, with inorganic sources of all other nutrient requirements Auxotroph
A species or genetic strain requiring one or more complex organic nutrients (such as amino acids, nucleotide bases, or enzymatic cofactors) for growth Microbiological Media Liquid (broth) vs. semisolid media Liquid medium Components are dissolved in water and sterilized
Semisolid medium A medium to which has been added a gelling agent Agar (most commonly used) Gelatin Silica gel (used when a non-organic gelling agent is required) Microbiological Media Chemically defined vs. complex media Chemically defined media
The exact chemical composition is known e.g. minimal media used in bacterial genetics experiments Complex media Exact chemical composition is not known Often consist of plant or animal extracts, such as soybean meal, milk protein, etc. Include most routine laboratory media,
e.g., tryptic soy broth Microbiological Media Selective media Contain agents that inhibit the growth of certain bacteria while permitting the growth of others Frequently used to isolate specific organisms from a large population of contaminants Differential media
Contain indicators that react differently with different organisms (for example, producing colonies with different colors) Used in identifying specific organisms Growth in Batch Culture Growth is generally used to refer to the acquisition of biomass leading to cell division, or reproduction A batch culture is a closed system in broth medium in which no additional nutrient is added
after inoculation of the broth. Growth in Batch Culture Typically, a batch culture passes through four distinct stages: Lag stage Logarithmic (exponential) growth Stationary stage Death stage Growth in Batch Culture
Mean Generation Time and Growth Rate The mean generation time (doubling time) is the amount of time required for the concentration of cells to double during the log stage. It is expressed in units of minutes. 1 Growth rate (min-1) = mean generation time Mean generation time can be determined directly from a semilog plot of bacterial concentration vs
time after inoculation Mean Generation Time and Growth Rate Mean Generation Time and Growth Rate Growth of E. coli 23716, 9-20-01 batch culture y = 0.0187e 0.0069x 2 R = 0.9928 10
Calibrated Petroff-Hausser counting chamber, similar to hemacytometer, can be used Generally very difficult for bacteria since cells tend to move in and out of counting field Can be useful for organisms that cant be cultured Special stains (e.g. serological stains or stains for viable cells) can be used for specific purposes Serial dilution and colony counting Also know as viable cell counts Concentrated samples are diluted by serial dilution
Measurement of Microbial Growth Serial dilution and colony counting Also know as viable cell counts Concentrated samples are diluted by serial dilution The diluted samples can be either plated by spread plating or by pour plating Measurement of Microbial Growth
Turbidity Based on the diffraction or scattering of light by bacteria in a broth culture Light scattering is measured as optical absorbance in a spectrophotometer Optical absorbance is directly proportional to the concentration of bacteria in the suspension Measurement of Microbial Growth Mass determination
Cells are removed from a broth culture by centrifugation and weighed to determine the wet mass. The cells can be dried out and weighed to determine the dry mass. Measurement of enzymatic activity or other cell components Growth in Continuous Culture
A continuous culture is an open system in which fresh media is continuously added to the culture at a constant rate, and old broth is removed at the same rate. This method is accomplished in a device called a chemostat. Typically, the concentration of cells will reach an equilibrium level that remains constant as long as the nutrient feed is maintained. Basic Chemostat System Our Chemostat System Factors that Influence Growth
Growth vs. Tolerance Growth is generally used to refer to the acquisition of biomass leading to cell division, or reproduction Many microbes can survive under conditions in which they cannot grow The suffix -phile is often used to describe conditions permitting growth, whereas the term tolerant describes conditions in which the organisms survive, but dont necessarily grow For example, a thermophilic bacterium grows under
conditions of elevated temperature, while a thermotolerant bacterium survives elevated temperature, but grows at a lower temperature Factors that Influence Growth Obligate (strict) vs. facultative Obligate (or strict) means that a given condition is required for growth Facultative means that the organism can grow under the condition, but doesnt require it
The term facultative is often applied to sub-optimal conditions For example, an obligate thermophile requires elevated temperatures for growth, while a facultative thermophile may grow in either elevated temperatures or lower temperatures Factors that Influence Growth Temperature
Most bacteria grow throughout a range of approximately 20 Celsius degrees, with the maximum growth rate at a certain optimum temperature Psychrophiles: Grows well at 0C; optimally between 0C 15C Psychrotrophs: Can grow at 0 10C; optimum between 20 30C and maximum around 35C Mesophiles: Optimum around 20 45C Moderate thermophiles: Optimum around 55 65 C Extreme thermophiles (Hyperthermophiles): Optimum around 80 113 C Factors that Influence Growth
pH Acidophiles: Neutrophiles Grow optimally between ~pH 0 and 5.5 Growoptimally between pH 5.5 and 8 Alkalophiles
Grow optimally between pH 8 11.5 Factors that Influence Growth Salt concentration Halophiles require elevated salt concentrations to grow; often require 0.2 M ionic strength or greater and may some may grow at 1 M or greater; example, Halobacterium Osmotolerant (halotolerant) organisms grow over a wide range of salt concentrations or ionic strengths;
for example, Staphylococcus aureus Factors that Influence Growth Oxygen concentration Strict aerobes: Require oxygen for growth (~20%) Strict anaerobes: Grow in the absence of oxygen; cannot grow in the presence of oxygen Facultative anaerobes: Grow best in the presence of
oxygen, but are able to grow (at reduced rates) in the absence of oxygen Aerotolerant anaerobes: Can grow equally well in the presence or absence of oxygen Microaerophiles: Require reduced concentrations of oxygen (~2 10%) for growth
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