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• Microbial metabolism is the main topic of this study session
• The specific topics covered are culturing microbes, measuring microbial growth, microbial growth dynamics, and environmental factors affecting growth, which includes temperature, pH, osmolarity, and oxygen

Cell nutrition

• Microbial growth refers to the increase in population size due to cell division
• Macronutrients are needed in large amounts
• Micronutrients are needed in very small amounts
• An average bacterial cell weighs about 10^-12g, and 75% of the mass is water
• The dry weight of a bacterial cell is 184^-15g
• Carbon, oxygen, nitrogen, hydrogen, phosphorus, and sulfur make up about 96% of the dry cell mass
• The next 3.7% of a cell's mass is potassium, sodium, calcium, magnesium, chlorine, and iron
• E. coli dry weight composition: 50% Carbon, 20% Oxygen, 14% Nitrogen, 8% Hydrogen, 3% Phosphorus, 1% Sulfur, Potassium, Sodium, 0.5% Calcium, Magnesium, Chlorine, 0.2% Iron, and 0.3% Other
• Trace elements, which are micronutrients, include metals needed in small amounts to perform essential biological funcitons
• Iron is used in hemoglobin cytochromes and respiratory enzymes
• Cobalt is used in Vitamin B12
• Copper is used in Repiration, cytochrome oxidase, superoxidase dismutase
• Manganese is used in superoxidase dismutase, carbohydrate metabolism
• Vitamins, amino acids, and purines are organic micronutrients

Growth media and laboratory culture

• Culture medium is a nutrient solution needed for growing a laboratory culture of a specific microorganism
• Defined media have an exact chemical composition (e.g., minimal media), often for genetic studies
• Complex media contain unknown quantities of nutrients, and are derived from natural sources like yeast extract or peptone like nutrient agar, tryptic soy broth
• Selective media inhibit unwanted organisms while allowing the target organism to grow such as agar for Salmonella
• Differential media is used to distinguish between organisms based on specific biochemical reactions like blood agar for hemolysis
• Enrichment media has additional nutrients to support the growth of fastidious organisms

Laboratory culture types

• Liquid broths are media used for growing large numbers of organisms
• Solid media has agar used to solidify the medium for isolating and observing colonies
• Semi-solid media contains lower agar concentrations, and are useful for motility studies
• Colonies are visible clusters of microorganism that comes from a single microbial cell
• Plates inoculated from mixed cultures such as a natural sample, or from contaminated pure cultures typically contain more than one colony type

Assessing microbial numbers

• Microbial numbers can be assessed by microscopic counts, viable counting and turbidimetric measures
• Microscopic count is the total cell count is used to enumerate cells with a microscope
• Viable counting is done with spread-plates and pour-plates
• Turbidimetric measures are done through optical density measuring

Microscopic counts of microbial cell numbers

• Total cell counts that enumerate the cells present can be done with a microscope
• You add sample, and ensure that there is not overflow between coverslip and the slide so it is at 0.02mm
• Whole grid has 25 large squares
• A large square has 16 small squares
• In practice, several large squares are counted and the numbers are averaged

Viable counting of microbial cell numbers

• Spread-Plate method involves spreading a volume of diluted culture over an agar plate which helps viable cells grow and yield single colonies
• Pour-plate method involves pipetting a known volume of media into a petri plate which helps molten agar medium added and mixed before allowing solidification
• One cell should form a single colony
• Platesshould contain between 30 and 300 colonies
• Colony Forming Units (CFUs) are individual/groups of cells capable of forming a visible colony on a solid growth medium under specific conditions

Limitations

• Direct microscopic counts usually reveal far microbes than are recoverable
• Some issues are bacteria with a natural chain length, cell clumping, biofilm formation

Turbidimetric measures of microbial cell numbers

• Cell scatter light, which makes the suspension look "cloudy" or turbid
• As the number of cells increases so does turbidity, resulting in light being scattered
• This can be measured with a spectrophotometer

Dynamics of microbial growth

• Microbial growth is the result of cell division
• Binary fission involves a cell enlongating before splitting in to two
• Generation time is the time required for population of microbial cells to double, and can take between 10 minutes to months
• Batch culture, is a fixed closed system where nutrients are limited
• Microbial growth curve is composed of four phases: lag, exponential, stationary, and decline

Microbial growth phases

• Lag phase: the period prior to the onset of growth
• Exponential phase: the period when the growing cell population doubles at regular intervals
• Stationary phase: period where the rate of growth and death are the same
• Decline phase: period where the act of cell death exceeds the rate of cell growth
• Important formula is: N(t) = N(0)2^n, where t is time, n is the number of generations during exponential growth.

Chemostat

• A chemostat is a continuous culture device that enables control over both the specific growth rate and growth yield of a microbial culture

Biofilms

• Biofilms are a population of cells enmeshed in a polysaccharide matrix that is attached to a surface
• Biofilm formation begins with the attachment of planktonic cells mediated by flagella, fimbriae, or pili
• Colonization starts when microbes begin to grow and secrete extracellular polysaccharides
• Development in biofilms involves cells changing their metabolisim
• Maturation leads to mushroom like channels and columns
• Also, metabolic differentiation of microbes occurs
• Dispersal involves cells leaving and colonizing new locations
• Biofilms are often associated with chronic infections which increase resistance to antimicrobial treatments
• Biofilms are used in bioreactors in order to break down organic pollutants and remove contaminants

Environmental effects on growth: Temperature

• Cardinal temperatures are the minimum, maximum, and optimum growth temperatures for a given organism
• Enzymatic reactions occur at increasingly rapid rates
• Enzymatic reactions occur at maximal possible rates when optimum
• When at minimum membrane gelling and transport processes slow down or stop
• When maximum, protein denaturing and collapse occur

Temperature Classes

• Psychrophiles thrive at low temperatures and optimal temperatures less than 15°C
• Mesophiles like moderate temperatures like Escherichia coli , with an optimum of 39°C
• Thermophiles thrives at high temperatures like Geobacillus stearothermophilus, with an optimum of 60°C
• Hyperthermophiles thrives at very high temperatures like Thermococcus celer, with an optimum of 88°C
• Hyperthermophile thrives at extreme temperatures like Pyrolobus fumarii, with an optimum of 106°C
• Molecular adaptations to life in the cold include cold-active enzymes having more a-helix structures, greater polar amino acids and membranes having greater longer chain length

pH

• Acidophiles thrive in acidic enviroments with example Picrophilus oshimae grows optimality at pH 0.7 and 60°
• Above pH 4, cells of P. oshimae spontaneuosly lyse
• Neutrophiles thrive at a neutral pH which ranges from 5.5 to 8
• Alkaliphiles thrive at a pH where there is higher alkalinity
• At pH adaptation includes Sodium (Na+) rather than H+ to drive tranpsort reactions and neutral cytoplasm

Osmolarity

• Marine micromorganisms accumulate solutes in their cells
• Compatible solutes are highly water-soluble organic molecules
• This includes sugars, alcohols, and amino acid derivatives which is widely disturbed among halopholic bacteria

Microbes and varying 02 environments

• Aerobes use O2 for respiration
• Anaerobes cannot use O2 and are typically inhibited by it
• Microaerophiles use O2 if levels are reduced from that in air
• Aerotolerant anaerobes cannot respire 02 but survive
• Obligate aerobes use O2 only when present at reduced levels
• Reactive Oxygen Species/ROS are reactive by-products that harm cells that cannot detoxify them.
• ROS scavenging enzymes include catalase, peroxidase, superoxide dismutase and reductase that destroy ROS.