Population Growth Cases in Stationary Phases | BIO 226N, Study notes of Biology

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Chapter 6

Microbial Growth

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Growth

• increase in cellular constituents that may result in: - increase in cell number - e.g., when microorganisms reproduce by budding or binary fission - increase in cell size - e.g., coenocytic microorganisms have nuclear divisions that are not accompanied by cell divisions
• microbiologists usually study population growth rather than growth of individual cells

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The Growth Curve

• observed when microorganisms are

cultivated in batch culture

• culture incubated in a closed vessel with a single batch of medium
• usually plotted as logarithm of cell

number versus time

• usually has four distinct phases

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Figure 6.

no increase

maximal rate of division and population growth

population growth ceases

decline in population size

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Lag Phase

• cell synthesizing new components
  • e.g., to replenish spent materials
  • e.g., to adapt to new medium or other conditions
• varies in length
  • in some cases can be very short or even absent

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Exponential Phase

• also called log phase
• rate of growth is constant
• population is most uniform in terms

of chemical and physical properties

during this phase

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cells are dividing and doubling in number at regular intervals

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8 Figure 6.

each individual cell divides at a slightly different time

curve rises smoothly rather than as discrete steps

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Balanced growth

• during log phase, cells exhibit

balanced growth

• cellular constituents manufactured at constant rates relative to each other

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Unbalanced growth

• rates of synthesis of cell components

vary relative to each other

• occurs under a variety of conditions
  • change in nutrient levels
    • shift-up (poor medium to rich medium)
    • shift-down (rich medium to poor medium)
  • change in environmental conditions

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Effect of nutrient

concentration on growth

Figure 6.

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Stationary Phase

• total number of viable cells remains

constant

• may occur because metabolically active cells stop reproducing
• may occur because reproductive rate is balanced by death rate

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Possible reasons for entry

into stationary phase

• nutrient limitation
• limited oxygen availability
• toxic waste accumulation
• critical population density reached

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Starvation responses

• morphological changes
  • e.g., endospore formation
• decrease in size, protoplast

shrinkage, and nucleoid

condensation

• production of starvation proteins
• long-term survival
• increased virulence

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Death Phase

• cells dying, usually at exponential

rate

• death
  • irreversible loss of ability to reproduce
• in some cases, death rate slows due

to accumulation of resistant cells

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The Mathematics of Growth

• generation (doubling) time
  • time required for the population to double in size
• mean growth rate constant
  • number of generations per unit time
  • usually expressed as generations per hour

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17 Figure 6.

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Measurement of

Microbial Growth

• can measure changes in number of

cells in a population

• can measure changes in mass of

population

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Measurement of Cell Numbers

• Direct cell counts
  • counting chambers
  • electronic counters
  • on membrane filters
• Viable cell counts
  • plating methods
  • membrane filtration methods

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Counting chambers

• easy, inexpensive, and quick
• useful for counting both eucaryotes and procaryotes
• cannot distinguish living from dead cells (^) Figure 6.

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Electronic counters

• microbial suspension forced through

small orifice

• movement of microbe through

orifice impacts electric current that

flows through orifice

• instances of disruption of current

are counted

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Electronic counters…

• cannot distinguish living from dead

cells

• quick and easy to use
• useful for large microorganisms and

blood cells, but not procaryotes

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Direct counts on membrane

filters

• cells filtered through special

membrane that provides dark

background for observing cells

• cells are stained with fluorescent

dyes

• useful for counting bacteria
• with certain dyes, can distinguish

living from dead cells

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Plating methods

• measure number of viable cells
• population size is expressed as colony forming units (CFU)

plate dilutions of population on suitable solid medium ↓ count number of colonies ↓ calculate number of cells in population

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Plating methods…

• simple and sensitive
• widely used for viable counts of

microorganisms in food, water, and

soil

• inaccurate results obtained if cells

clump together

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Membrane filtration

methods

Figure 6.6 (^) especially useful for analyzing aquatic samples

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Measurement of Cell Mass

• dry weight
  • time consuming and not very sensitive
• quantity of a particular cell constituent
  • e.g., protein, DNA, ATP, or chlorophyll
  • useful if amount of substance in each cell is constant
• turbidometric measures (light scattering)
  • quick, easy, and sensitive

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29 Figure 6.

more cells ↓ more light scattered ↓ less light detected

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The Continuous Culture of

Microorganisms

• growth in an open system
  • continual provision of nutrients
  • continual removal of wastes
• maintains cells in log phase at a

constant biomass concentration for

extended periods

• achieved using a continuous culture

system

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The Chemostat

• rate of incoming medium = rate of removal of medium from vessel
• an essential nutrient is in limiting quantities Figure 6.

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Dilution rate and microbial

growth

Figure 6.

dilution rate – rate at which medium flows through vessel relative to vessel size

note: cell density maintained at wide range of dilution rates and chemostat operates best at low dilution rate

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The Turbidostat

• regulates the flow rate of media

through vessel to maintain a

predetermined turbidity or cell

density

• dilution rate varies
• no limiting nutrient
• turbidostat operates best at high

dilution rates

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Importance of continuous

culture methods

• constant supply of cells in exponential phase growing at a known rate
• study of microbial growth at very low nutrient concentrations, close to those present in natural environment
• study of interactions of microbes under conditions resembling those in aquatic environments
• food and industrial microbiology

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The Influence of

Environmental Factors on

Growth

• most organisms grow in fairly

moderate environmental conditions

• extremophiles
  • grow under harsh conditions that would kill most other organisms

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Solutes and Water Activity

• water activity (aw )
  • amount of water available to organisms
  • reduced by interaction with solute molecules (osmotic effect) higher [solute] ⇒ lower aw
  • reduced by adsorption to surfaces (matric effect)

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Osmotolerant organisms

• grow over wide ranges of water activity
• many use compatible solutes to increase their internal osmotic concentration - solutes that are compatible with metabolism and growth
• some have proteins and membranes that require high solute concentrations for stability and activity

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Effects of NaCl on microbial

growth

• halophiles
  • grow optimally at >0.2 M
• extreme halophiles
  • require >2 M

Figure 6.

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pH

• negative logarithm of the hydrogen ion concentration

Figure 6.

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pH

• acidophiles
  • growth optimum between pH 0 and pH 5.
• neutrophiles
  • growth optimum between pH 5.5 and pH 7
• alkalophiles
  • growth optimum between pH8.5 and pH 11.

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pH

• most acidophiles and alkalophiles maintain an internal pH near neutrality - some use proton/ion exchange mechanisms to do so
• some synthesize proteins that provide protection - e.g., acid-shock proteins
• many microorganisms change pH of their habitat by producing acidic or basic waste products - most media contain buffers to prevent growth inhibition

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Temperature

• organisms exhibit distinct cardinal growth temperatures - minimal - maximal - optimal

Figure 6.

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Figure 6.

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Adaptations of thermophiles

• protein structure stabilized by a variety of means - e.g., more H bonds - e.g., more proline - e.g., chaperones
• histone-like proteins stabilize DNA
• membrane stabilized by variety of means
  • e.g., more saturated, more branched and higher molecular weight lipids
  • e.g., ether linkages (archaeal membranes)

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Oxygen Concentration

Figure 6.

need oxygen

prefer oxygen

ignore oxygen oxygen is toxic

< 2 – 10% oxygen

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Basis of different oxygen

sensitivities

• oxygen easily reduced to toxic products
  • superoxide radical
  • hydrogen peroxide
  • hydroxyl radical
• aerobes produce protective enzymes
  • superoxide dismutase (SOD)
  • catalase

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Figure 6.

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Pressure

• barotolerant organisms
  • adversely affected by increased pressure, but not as severely as nontolerant organisms
• barophilic organisms
  • require or grow more rapidly in the presence of increased pressure

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Radiation

Figure 6.

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Radiation damage

• ionizing radiation
  • x rays and gamma rays
  • mutations → death
  • disrupts chemical structure of many molecules, including DNA - damage may be repaired by DNA repair mechanisms

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Radiation damage…

• ultraviolet (UV) radiation
  • mutations → death
  • causes formation of thymine dimers in DNA
  • DNA damage can be repaired by two mechanisms - photoreactivation – dimers split in presence of light - dark reactivation – dimers excised and replaced in absence of light

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Radiation damage…

• visible light
  • at high intensities generates singlet oxygen ( 1 O 2 ) - powerful oxidizing agent
  • carotenoid pigments
    • protect many light-exposed microorganisms from photooxidation

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Microbial Growth in Natural

Environments

• microbial environments are

complex, constantly changing, and

may expose a microorganism to

overlapping gradients of nutrients

and environmental factors

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Growth Limitation by

Environmental Factors

• Leibig’s law of the minimum
  • total biomass of organism determined by nutrient present at lowest concentration
• Shelford’s law of tolerance
  • above or below certain environmental limits, a microorganism will not grow, regardless of the nutrient supply

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Responses to low nutrient

levels

• oligotrophic environments
• morphological changes to increase

surface area and ability to absorb

nutrients

• mechanisms to sequester certain

nutrients

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Counting Viable but

Nonculturable Vegetative

Procaryotes

• stressed microorganisms can temporarily lose ability to grow using normal cultivation methods
• microscopic and isotopic methods for counting viable but nonculturable cells have been developed

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Quorum Sensing and

Microbial Populations

• quorum sensing
  • microbial communication and cooperation
  • involves secretion and detection of chemical signals

Figure 6.

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Processes sensitive to

quorum sensing: gram-

negative bacteria

• bioluminescence ( Vibrio fischeri )
• synthesis and release of virulence factors ( Pseudomonas aeruginosa )
• conjugation ( Agrobacterium tumefaciens )
• antibiotic production ( Erwinia carotovora, Pseudomonas aureofaciens )
• biofilm production ( P. aeruginosa )

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Quorum sensing: gram-

positive bacteria

• often mediated by oligopeptide pheromone
• processes impacted by quorum sensing:
  • mating ( Enterococcus faecalis )
  • transformation competence ( Streptococcus pneumoniae )
  • sporulation ( Bacillus subtilis )
  • production of virulence factors ( Staphylococcus aureus )
  • development of aerial mycelia ( Streptomyces griseus )
  • antibiotic production ( S. griseus )