Cell Growth and Binary Fission Growth: increase in the number of cells Binary fission: cell elongation following enlargement of a cell to twice its minimum size Generation time: time required for a population of microbial cells to double During cell division each daughter cell receives a chromosome and sufficient copies of all other cell constituents to exist as an independent cell

Binary Fission in a Rod-Shaped Prokaryote Figure 6.1

Growth Terminology and the Concept of Exponential Growth Most bacteria have shorter generation times than eukaryotic microbes Generation time is dependent on growth medium and incubation conditions Exponential growth: growth of a microbial population in which cell numbers double within a specific time interval During exponential growth, the increase in cell number is initially slow but increases at a faster rate

The Rate of Growth of a Microbial Culture Figure 6.8

Calculating Microbial Growth Parameters Figure 6.9

The Mathematics of Exponential Growth Increase in cell number in an exponentially growing bacterial culture is a geometric progression of the number 2 Relationship exists between the initial number of cells present in a culture and the number present after a period of exponential growth: N = No2n where N is the final cell number, No is the initial cell number, and n is the number of generations during the period of exponential growth

The Mathematics of Exponential Growth Generation time (g) of the exponentially growing population is g = t/n where t is the duration of exponential growth and n is the number of generations during the period of exponential growth

The Mathematics of Exponential Growth Specific growth rate (k) is calculated as k = 0.301/g Division rate (v) is calculated as v = 1/g

The Microbial Growth Cycle Batch culture: a closed-system microbial culture of fixed volume

Continuous Culture: The Chemostat Continuous culture: an open-system microbial culture of fixed volume Chemostat: most common type of continuous culture device Both growth rate and population density of culture can be controlled independently and simultaneously Dilution rate: rate at which fresh medium is pumped in and spent medium pumped out Concentration of a limiting nutrient

Schematic for Continuous Culture Device (Chemostat) Figure 6.11

Continuous Culture: The Chemostat In a chemostat The growth rate is controlled by dilution rate The growth yield (cell number/ml) is controlled by the concentration of the limiting nutrient In a batch culture growth conditions are constantly changing; it is impossible to independently control both growth parameters

The Effect of Nutrients on Growth Figure 6.12

Continuous Culture: The Chemostat Chemostat cultures are sensitive to the dilution rate and limiting nutrient concentration Too high a dilution rate, the organism is washed out Too low a dilution rate, the cells may die from starvation Increasing limiting nutrient concentration results in greater biomass but same growth rate

Steady-State Relationships in the Chemostat Figure 6.13

Measurements of Total Cell Numbers: Microscopic Counts Microbial cells can be enumerated by microscopic observations; simple but results can be unreliable Limitations of microscopic counts Cannot distinguish between live and dead cells without special stains Small cells difficult to see and can be overlooked Precision is difficult to achieve A phase-contrast microscope is required if a stain is not used

Measurements of Total Cell Numbers: Microscopic Counts Limitations of microscopic counts (cont’d) Cell suspensions of low density (< 106 cells/ml) hard to count Motile cells need to immobilized Debris in sample can be mistaken for cells A second method for enumerating cells in liquid samples is with a flow cytometer

Direct Microscopic Counting Procedure Figure 6.14

Viable Cell Counting Viable cell counts (plate counts): measurement of living, reproducing population Two main ways to perform plate counts Spread-plate method Pour-plate method To obtain the appropriate colony number, the sample to be counted should always be diluted

Spread-Plate Method for the Viable Count Figure 6.15

Pour-Plate Method for the Viable Count Figure 6.15

Procedure for Viable Counting Using Serial Dilutions Figure 6.16

Viable Cell Counting Plate counts can be highly unreliable when used to assess total cell numbers of natural samples (e.g., soil and water) The Great Plate Anomaly: direct microscopic counts of natural samples typically reveal far more organisms than are recoverable on plates of any given culture medium

Viable Cell Counting Why is this? Microscopic methods count dead cells whereas viable methods do not Different organisms in even a very small sample may have vastly different requirements for resources and conditions in laboratory culture

Measurements of Microbial Mass: Turbidimetric Methods Turbidity measurements are an indirect but very rapid and useful method of measuring microbial growth Most often measured with a spectrophotometer and measurement referred to as optical density (O.D.)

Turbidity Measurements of Microbial Growth Figure 6.17a

Turbidity Measurements of Microbial Growth To relate a direct cell count to a turbidity value, a standard curve must first be established Figure 6.17b

Measurements of Microbial Mass: Turbidimetric Methods Turbidity measurements Quick and easy to perform Typically do not require destruction or significant disturbance of sample Sometimes problematic (e.g., microbes that form clumps or biofilms in liquid medium)

Effects of Temperature on Microbial Growth Microorganisms can be classified into groups by their growth temperature optima Psychrophile: low temperature Mesophile: midrange temperature Thermophile: high temperature Hyperthermophile: very high temperature

Temperature and Growth Relations in Different Classes Figure 6.19

Effects of Temperature on Microbial Growth Mesophiles: organisms that have midrange temperature optima; found in Warm-blooded animals Terrestrial and aquatic environments Temperate and tropical latitudes

Microbial Growth at Cold Temperatures Extremophiles Organisms that have evolved to grow optimally under very hot or very cold conditions Psychrophiles Organisms with cold temperature optima; the most extreme representatives inhabit permanently cold environments Psychrotolerant Organisms that can grow at 0ºC but have optima of 20ºC to 40ºC; more widely distributed in nature than psychrophiles

Antarctic Microbial Habitats and Microorganisms Figure 6.20a and b

Microbial Growth at Cold Temperatures Molecular Adaptations to Psychrophily Production of enzymes that function optimally in the cold; features that may provide more flexibility More of -helices than -sheets More polar and less hydrophobic amino acids Fewer weak bonds Decreased interactions between protein domains

Microbial Growth at Cold Temperatures Molecular Adaptations to Psychrophily (cont’d) Transport processes function optimally at low temperatures due to modifications of cytoplasmic membranes High unsaturated fatty acid content

Microbial Growth at High Temperatures Above ~65°C only prokaryotic life forms exist Thermophiles: organisms with growth temperature optima between 45ºC and 80ºC Hyperthermophiles: organisms with optima greater than 80°C Inhabit hot environments including boiling hot springs and seafloor hydrothermal vents that can have temperatures in excess of 100ºC

Upper Temperature Limits for Growth of Living Organisms

Microbial Growth at High Temperatures Hyperthermophiles in Hot Springs Chemoorganotrophic and chemolithotrophic species present High prokaryotic diversity (both Archaea and Bacteria represented) Thermal gradients form along edges of hot environments Distribution of microbial species along the gradient is dictated by organism’s biology

Growth of Hyperthermophiles in Boiling Water Figure 6.22

Growth of Thermophilic Cyanobacteria in a Hot Spring Figure 6.23

Microbial Growth at High Temperatures Studies of thermal habitats have revealed Prokaryotes are able to grow at higher temperatures than eukaryotes Organisms with the highest temperature optima are Archaea Nonphototrophic organisms can grow at higher temperatures than phototrophic organisms

Microbial Growth at High Temperature Molecular Adaptations to Thermophily Enzyme and proteins function optimally at high temperatures; features that provide thermal stability Critical amino acid substitutions in a few locations provide more heat-tolerant folds An increased number of ionic bonds between basic and acidic amino acids resist unfolding in the aqueous cytoplasm Production of solutes (e.g., di-inositol phophate, diglyercol phosphate) help stabilize proteins

Microbial Growth at High Temperature Hyperthermophiles and produce enzymes widely used in industrial microbiology E.g., Taq polymerase; used to automate the repetitive steps in the polymerase chain reaction (PCR) technique

Microbial Growth at Low or High pH The pH of an environment greatly affects microbial growth Some organisms have evolved to grow best at low or high pH, but most organisms grow best between pH 6 and 8 (neutrophiles)

The pH Scale Figure 6.24

Microbial Growth at Low or High pH Acidophiles: organisms that grow best at low pH (< 6) Some obligate acidophiles; membranes destroyed at neutral pH Stability of cytoplasmic membrane critical Alkaliphiles: organisms that grow best at high pH (> 9) Some have sodium motive force rather than proton motive force

Microbial Growth at Low or High pH The internal pH of a cell must stay relatively close to neutral even though the external pH is highly acidic or basic Internal pH has been found to be as low as 4.6 and high as 9.5 in extreme acido- and alkaliphiles, respectively

Oxygen and Microbial Growth Aerobes: require oxygen to live Anaerobes: do not require oxygen and may even be killed by exposure Facultative organisms: can live with or without oxygen Aerotolerant anaerobes: can tolerate oxygen and grow in its presence even though they cannot use it Microaerophiles: can use oxygen only when it is present at levels reduced from that in air

Growth Versus Oxygen Concentration Figure 6.27

Oxygen Relationships of Microorganisms

Incubation under Anoxic Conditions