Microbial Growth

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Microbial Growth
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Microbial Growth

• Increase in number of cells rather than size
• Growth of most microorganisms occurs by the
  process of binary fission
• DNA replication
• Double amount of macromolecules, monomers, and
  inorganic ions
• Growth of membrane and cell wall
• Division
• Generation time varies (Typical 1 - 3 hours)
• Dependent on nutritional and genetic factors
• E. coli 20 minutes to divide ? optimal
  conditions

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Figure 6.1
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Cell division and chromosome replication

• Regulated by Fts proteins (filamentous
  temperature sensitive)
• Essential for cell division in all prokaryotes
• Fts proteins interact to form a division
  apparatus in the cell called the divisome.
• FTSz
• Forms ring around center of cell
• Directs cell division at the central plane of
  cell
• ZipA
• Anchor that connects FtsZ ring to cytoplasmic
  membrane
• FtsA
• Helps connect FtsZ ring to membrane and also
  recruits other divisome proteins

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Figure 6.2
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6.2 - Fts Proteins and Cell Division

• DNA replicates before the FtsZ ring forms
• Location of FtsZ ring is facilitated by Min
  proteins
• Direct the placement of FTSz between 2 nucleoids
• FtsK protein mediates separation of chromosomes
  to daughter cells
• GTP
• Used as fuel source for FTSz polymerization/depoly
  merization

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Cell Division Cycle
DNA Replication
FtsZ depolymerization

• GTP as fuel
• Septum formation

FtsZ Ring formation

• Fueled by GTP
• Between 2 nucleoids
• Directed by Min

Cell Elongation
Divisome Formation

• Chromosomes pulled apart

• Fts divisome proteins
• New cell wall and membrane produced

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DNA Replication and Cell-Division Events
Figure 6.3
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6.4 - Peptidoglycan Synthesis and Cell Division

• Production of new cell wall material is a major
  feature of cell division
• In cocci, cell walls grow in opposite directions
  outward from the FtsZ ring
• In rod-shaped cells, growth occurs at several
  points along length of the cell

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Cell Wall Formation

• Preexisting peptidoglycan needs to be severed to
  allow newly synthesized peptidoglycan to form
• Begins at the FtsZ ring
• Autolysins (enzymes that are similar to lysozyme)
  breaks glycosidic bonds creating small openings

Figure 6.7a
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Cell Wall Formation

• New (M-G-pep) created in cytoplasm
• New cell wall material is added across the
  openings
• Bactoprenol?a hydrophobic alcohol that
  facilitates transport of new glycan units through
  the cytoplasmic membrane to become part of the
  growing cell wall
• Wall band junction between new and old
  peptidoglycan
• Glycolases
• A process of spontaneous cell lysis called
  autolysis can occur unless new cell wall
  precursors are spliced into existing
  peptidoglycan to prevent a breach in
  peptidoglycan integrity at the splice point.

Figure 6.7a
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Transpeptidation

• Final step in cell wall synthesis
• Form cross links between NAM in adjacent chains
  of peptidoglycan
• Inhibited by penicillin

Figure 6.7b
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Population Growth

• Growth rate change in cell number or cell mass
  of population
• A generation is the interval of two cells from
  one
• Generation time (doubling time)
• Time it takes to produce two new cells
• Time for cell mass or to double
• Varies greatly
• Type of organism
• Temperature
• Nutrients
• Other conditions
• Norm 1-3 hours
• Exponential growth (Log phase growth)
• When population doubles/ unit of time
• Lets take look at animation
• http//www.biology.arizona.edu/biomath/tutorials/A
  pplications/Population.html

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Bacteria grow exponentially
Most bacteria divide in a short amount of time
and produce a large amount of bacteria easier
to represent these large numbers by logarithmic
scales
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Plotting bacterial growth
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Growth Calculations

• If you start with 1 cell how many do you have
  after 4 generations?
• No initial number of cells
• N cells after n generations
• nnumber of generations
• Formula?N No(2n)
• N1(16)16 cells
• What if you start with 100 cells?
• What if you start with 100 cells and go for 5
  generations?

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Growth Calculations

• E. coli has a generation time of 20 minutes. If
  you start with 1 E. coli cell how many do you
  have after 2 hours?
• ggeneration time and ttime
• Formula?nt/g
• n(2 hours x 60minutes/hour)/20 minutes ?
• N No(2n)
• N1(26)64 cells
• 5 hours?
• N32,768 cells

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Plotting growth versus time The smaller the
generation time, the faster the growth. The
faster the growth, the greater the slope in the
line. g6 hours slope 0.05 g2 hours slope 0.15
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Realistic Growth Calculations

• How do you determine n if you know N and No only?
• You start with 2 cells and end up with 2,000
  after 2 hours so how many generations? What is
  generation time?
• n3.3(logN-logNo)
• So n3.3(log (2000) log (2))
• n3.3(3.3-0.3)9.9 generations
• gt/n
• g120 minutes/9.9 generations12.12 minutes per
  generation

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More Growth Calculations

• K is the growth rate constant or the number of
  generations per unit time for a given organism
  under a given set of conditions
• K is used to optimize growth conditions the
  faster the growth the larger the K
• Kln2/g
• Example
• Generation time 30 minutes (k0.023)
• Generation time 60 minutes (k0.011)

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Summary

• The faster the growth the
• greater the k (growth constant)
• greater the slope when plotting cell
  concentration per unit time
• smaller the g (generation time)

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Recall This Question Again

• E. coli has a generation time of 20 minutes. If
  you start with 1 E. coli cell how many do you
  have after 24 hours?
• We determined 4.72 x 1021 cells
• Theoretically this is correct if cells didnt
  die, run out of nutrients, sit in a pool of their
  own waste for several hours, etc.
• The growth calculations you learned pertain to
  EXPONENTIAL PHASE ONLY!

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Growth Cycle

• Lag phase time it takes for cell to start
  growing once inoculated
• Take in nutrients, synthesize essential
  components, repair damage, adjust to new
  media/nutrients, adjust to new concentration of
  nutrients
• Varies depending on conditions and nature of
  culture
• Exponential or log phase cells growing
  exponentially
• When population doubles/ unit of time
• Rate increases with each new generation
• Most metabolically active, but most sensitive
• Stationary phase No net increase or decrease in
  population
• Nutrients run out or waste build up
• Metabolism and biosynthesis still occurring
• Death phase cells lysing gt new cells

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Growth Curve
Plot log cell concentration over time Plot OD
versus time for comparison here We will learn
more about these counting methods in lab
Figure 6.10
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Continuous versus Batch

• Continuous
• Chemostat
• No growth phases
• Always exponential
• Flow system with constant volume
• Fresh media added as depleted media discarded
• Can control growth rate and population density
  independently
• Purpose Measure growth properties, physiology,
  microbial ecology

• Batch
• Test tube
• Distinct growth phases
• Fixed volume of media and no flow
• Media eventually depleted and no replacement
• Growth rate is dependent on population density
• Purpose growth overnight cultures.

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Figure 6.11
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Continuous Culture

• Growth Rate (GR)
• Increase in cell number per unit time
• Doubling time decreases as GR increases
• Growth Yield (GY)
• Number of cells present at a given time
• Cell concentration
• Nutrient concentration and dilution rate affects
  the growth rate and yield

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GR vs. GY

• Growth rate controlled independently from growth
  yield
• To increase GR increase dilution rate
• Yield stays generally the same
• To increase GY increase concentration of
  nutrients
• Rate stays generally the same
• Industrial microbiologists grow bacteria to
  obtain a lot of cells in a short amount of time

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As nutrient concentration increases the GY
increases but GR stays steady after steady state
reached.
Figure 6.12
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As dilution rate increases GR increases (doubling
time decreases). As dilution increases no change
in GY until a POINT!!!! Wash out Flow too
fast?washes culture out?diluted before they can
grow
Figure 6.13
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Applications

• Can control GR and GY independently
• Cells always in exponential phase
• Most physiological experiments require
  exponential phase
• Can determine nutrient effects on population or
  mimic natural environment
• By adjusting dilution rate and nutrient levels,
  the experimenter can obtain dilute, moderate and
  dense populations growing at slow, moderate or
  rapid growth rates

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Factors that affect bacterial growth

• Temperature
• pH
• Osmotic pressure/water availability
• Oxygen

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Temperature

• Cardinal temperatures
• Minimum growth temperature
• Lowest temperature at which an organism will grow
• Below this temp.?nutrient transport difficulty
  due to the fact that membrane gels and transport
  too slow
• Optimum growth temperature
• Temperature at which an organism grows best
• Metabolic enzyme reactions occurring at maximum
  rate
• Maximum growth temperature
• Highest temperature at which an organism will
  grow
• Above this temp.?protein denaturation membrane
  collapse, and lysis
• All can be modified slightly by other
  environmental properties
• Usually a 30º range (C) for prokaryotes
• Extremophiles live at extreme hot and cold
  temperatures

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The Cardinal Temperatures
Figure 6.18
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Temperature Classes

• Psychrophiles
• Cold lovers
• Optimum 0 -15 ºC (depends on organismusually
  around 4 ºC)
• RANGE -10 ºC ? 20 ºC (cannot survive at room
  temp!)
• Min is typically below zero
• Found in polar regions, at high altitudes, and in
  depths of oceans (constant cold)
• Algae in sea ice and snow fields
• Psychrotolerant (psychrotroph)
• Optimum 20 - 40 ºC
• RANGE 1 ºC ? 40 ºC
• Grows best at refrigerator temperatures, but can
  grow at low temperatures
• Typically cannot grow at freezing temps.
• Found in soils and water and foods in fridge
• Enzymes sensitive to heat b/c of structure
• Polar and Hydrophobic amino acids?increase
  flexibility
• More a helices and fewer ß sheets?increase
  flexibility
• Membranes well suited
• Increase in unsaturated fatty acids (more fluid)

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Psychrotrophs
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Temperature Classes

• Mesophiles
• Optimum 37-40 ºC (body temp)
• RANGE 12 ? 48 ºC
• Most common
• Most pathogens
• E. coli
• Thermophiles
• Heat loving
• Optimum 45-80 ºC (depending on organism)
• RANGE 40 ? 85ºC
• Compost, soils, hot water heaters, some hot
  springs
• Hyperthermophiles
• Optimum 90-121 ºC
• RANGE 89 ? 120 ºC
• Steam vents, hot springs, volcanoes
• Mostly Archaea
• Results of studies of different organisms
• Prokaryotes can grow at higher temps than
  Eukaryotes
• Most thermophiles (hyperthermophiles) are archaea

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Temperature Requirements
Figure 6.19
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How can thermophiles and hyperthermophiles thrive
at high temperatures?

• Enzymes more heat stable
• Only a few key amino acids are different from
  mesophiles
• Increase in salt bridges (ionic bonds) between
  amino acids
• Densely packed hydrophobic interiors
• Example of heat stable enzyme Taq polymerase
  used in PCR, isolated from Thermus aquaticus
• Membranes are more heat stable
• Bacteria - saturated fatty acids (dec. fluidity)
  and stronger hydrophobic environment (greater
  interaction of fatty acid tails)
• Archaea contain isoprene units?lipid monolayer
  and ether linkage

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Physical Requirements

• pH
• Most natural environments pH 5-9
• Most bacteria produce organic acids as they grow
  and metabolize
• When growing bacteria, pH can change during
  growth so buffers are added to moderate the pH
• pH should be near normal on inside of cell
• Acidophiles
• Grow at low pH (lt5)
• Fungi in general and some bacteria (obligate
  must grow at low pH)
• If pH is increased, membranes are destroyed and
  cells lyse
• Thiobacillus and acid mine drainage (pH 1)
• Alkaliphiles
• Grow at high pH (gt10-11 pH)
• Soda lakes, high carbonate soils

Figure 6.24
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Preserving Food

• Most bacteria grow best between pH 6.5 7.5
• Neutrophiles - pH 5.4 - 8.5
• Foods can be preserved by acid pH

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Osmotic Effects on Microbial Growth

• Osmosis
• Positive water balance
• Normally, cytoplasm has higher solute
  concentration than environment (positive water
  balance)
• Water activity (aw) vapor pressure of air to
  water
• Low aw hypertonic
• Hypotonic environments
• What happens?
• Plasmolysis
• Caused by hypertonic environments
• Use of salt as a preservative

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Salt Lovers

• Halophiles
• Specific requirement for Na
• Can grow at high salt concentration without
  negative water balance.
• Mild require 1-6 NaCl
• Moderate require 6-15 NaCl
• Extreme require 15-30
• Halotolerant can tolerate low aw, but not
  optimal for growth
• How can a cell exist in salty environment?
• Compatible solutes do not inhibit cell activity
• Increase in internal solute concentration
• Synthesis versus transport of a compatible solute

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Effect of salt concentrations on growth of
microorganisms
Figure 6.25
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Others

• Osmophiles
• Tolerates high sugar concentrations which cause
  low aw
• Xerophiles
• Tolerate dry environments

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Chemical Requirements

• Oxygen
• Variation in need to metabolize O2
• Divided into several groups
• Obligate (strict) aerobes
• Aerobic metabolism (requires O2 to make energy)
• Growth at 21 O2
• Detoxify products of metabolism
• Microaerophiles
• Aerobic metabolism (requires O2 in small amounts
  for energy)
• Growth at reduced O2 levels
• Facultative anaerobe (E. Coli)
• In presence of O2 uses aerobic metabolism to make
  energy (faster)
• In absence of O2 will ferment (less energy
  produced)
• Obligate (strict) anaerobe (Clostridium)
• Anaerobic metabolism or fermentation
• No O2 metabolism and killed by O2
• Aerotolerant
• Anaerobic metabolism or fermentation (no benefit
  from oxygen)
• No O2 metabolism, but tolerates O2

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Toxic Forms of Oxygen

• Products of O2 metabolism?toxic
• Singlet oxygen O2 boosted to a higher-energy
  state
• Superoxide free radicals O2
• Peroxide anion O22
• Hydroxyl radical (OH?)

Figure 6.29
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Toxic Forms of Oxygen

• Organisms that use aerobic metabolism must
  detoxify these products
• Catalase enzyme 2 H2O2?2 H2O O2
• Peroxidase enzyme H2O2?2 H H2O
• Superoxide dismutase enzyme detoxifies O2-and
  OH
• Obligate anaerobes lack these enzymes

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How are anaerobic organisms grown?

• They grow at the bottom of tubes, away from
  oxygen
• Reducing agents added to media of anaerobes
• Resazurin reduce O2 ? H2O
• Anaerobic jars and chambers (air replacement)

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Chemical Requirements

• Oxygen (O2)

• Thioglycollate media
• Which are aerobes, anaerobes, facultative
  anaerobes, microaerophiles, aerotolerant?

Figure 6.27