class 5 growth
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Microbial Growth
• A complex metabolic process involvedcatabolic and anabolic reactions.
• leads to cell division⇒a rise in cell number (1→2, 2→4….2n); an increase in the number
of cells• microorganisms reproduce by binary
fission or budding
• Measurement of growth normally followschange in the total cell number (or cell mass), not individual cell (too small toobserve)
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How does reproduction occur?
– Binary fission – asexual reproductive process• DNA replication Cell elongation Septum
formation Completion of septum w/ formation of
distinct walls cell separation and formation of
daughter cell• All types of molecules double in amount: protein,
DNA, RNA, lipids for membranes, cell wall materials,
small molecules. Everything is evenly distributed.
• All biosynthetic events must be carefully coordinated
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Microbial Growth
Yeast is an example of
microbe that reproduces by budding
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All cellcomponents
double.
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• Generation: 1 cell 2 cells, i.e.
population double.• Generation time (doubling time)
- the time required for a cell division
- depends on genetic or nutritional (growthmedium, incubation conditions….)
• Growth rate: change in cell number or cell
mass/time
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Time for reproduction
• Speed depends on:
– Species
– Conditions
• Bacteria proliferate very rapidly in a favorable
environment.
– E. coli can divide every 20 minutes,
producing a colony of 107
to 108
bacteria in aslittle as 12 hours.
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• Through binary fission, most of the bacteria
in a colony are genetically identical to the
parent cell. (clones -identical progeny
produced asexually)
• Short generation time -fast growth -more
mutation rate (over time) -more diversity
-better adaptation.
• Long generation time -slow growth.
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Growth Curve (Batch culture)
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Microbial Growtha. Lag phase
microbes are adjusting tothe new substrate (foodsource)
b. Exponentialgrowth phasemicrobes have acclimatedto the conditions, celldivided with μ
maxat any
particular period
c. Stationary phase limiting substrate or electron acceptor limitsthe growth rate
d. Decay phase
substrate supply has beenexhausted
Time
log [X ]
32 41
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During Exponential growth
N = N0 x 2n
N = final cell numbers (cell mass)
N0 = initial cell numbers (cell mass)
n = number of generations
g (generation time) = t / n
t: time of exponential growth
Experimental data
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N = N0 * 2n
logN = logN0 + n log2
n = (logN - logN0)/log2
= (logN - logN0)/0.301
= 3.3(logN - logN0)
Mean generation time(g) = total growth time(t)/ n
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Exponential Phase
• Log phase growth is first order, ie.,
Growth rate ∝ to population size
•lnX vs. t is linear, slope = µ
µ units are 1/t (i.e. hr -1)
Xdt
dX µ=
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Growth rate constant (Specific growth
rate)
• μ = number of generations occurred per unit timeduring exponential growth (unit: time-1),
• μ = n/t
when Nt=2N0 ,
n = (logN - logN0)/0.301, t=g
⇒ μ = n/t =log2N0-logN0 /0.301*g
= 1/g (reciprocal of generation time, g)
G th t t t (S ifi th t ) i
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- during exponential growth, the concentration of biomass, X,increases as a function of time due to conversion of food tobiomass:
dX
dt
dX
X
⇒ lnX = μ t + lnXo
⇒ lnX vs. t 做圖
X = cell number or cell mass
μ is determined from the linear portion of a semilog plot of growthversus time
= μX
= μdt
Growth rate constant (Specific growth rate) in a
batch culture
μ is the specific growth rate constant (d-1). This
represents the mass of cells produced/mass of cells
per unit of time
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Effect of [Substrate] on growth rate constant
• 1940s, Monod
µ max S
Ks + S
µ : specific grwoth rate (1/time)
µ max : maximum specific growth rate constant
S: substrate concentration (mass/vol.)
Ks: half-saturation constant (mass/vol.) constant
• Ks determines how rapidly µ approaches µ max
The small it is, the lower the substrate concentration at which µ
approaches µ max .
µ
=
Monod equation
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Monod Growth Kinetics
SK
S
s
max
+
µ=µ
•Relates specific growth rate, μ , to substrate
concentration, S
•Empirical!---no theoretical basis—it just
“fits”!•Have to determine μ max and K s in the lab
•Each μ is determined for a different starting S
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@ low [substrate]⇒ µ ∝ [S], first-order reaction
@ high [substrate],⇒ µ = µ max , constant, zero-order reaction
the rate at which the susbtrate concentration is not limiting
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µ =µ max ⇒zero
order
µ ∝ [S]⇒first
order
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Continuous Culture
• A flow system of adding constant volume of fresh medium continuously and removing
spent medium continuously at a constant rate
• keep culture in a constant environment⇒cell number and nutrient status are
constant
⇒ steady state• Chemostat 恆化器
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Steady-state relationships in the chemostat
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Steady-state relationships in the chemostat
Measurement of growth
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Measurement of growth
Direct Measurement
• Change in cell numbers or other cell mass (protein, nucleic acid), or total cell dry weight
(1). Direct count (Total cell count)
Petroff-Hausser counting chamber (bacteria)Coulter counter (protozoa, algae, yeast)
(2). Viable count: count viable cells onlycount colonies on plate (Plate count)
unit: CFU (Colony Forming Units)
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Direct microscopic counting
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Limitation of direct count
• Both living cell dead cell count• Too small cells easily miss
• Less precision
• Necessity of staining or need a phasemicroscope
• Hard to see cells of low density population
• Motile cells hard to count, need toimmobilized first
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Viable count (I)
Vi bl
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Indirect Measurement
• cell number is estimated by turbidity
measurement
• Determine amount of light scattered by a
suspension of cells with a photometer or
spectrophotometer
• Common used wavelengths: 540 nm, 600 nm,
or 660 nm and express as optical density (OD
540 nm)
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Temperature
• Has the major effect on microbial growth• raising the temperature increases the
reaction rate:
1. rate of enzyme-catalyzed reaction
2. rate of diffusion of substrate into cell.
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Temperature
- based on optimal growth temperature (T opt )
Psychrophiles: below 15oC, Tmax< 20℃
Mesophiles: 20 – 45oC
Thermophiles: above 45oC
Thermophile: 45 – 60oC
Extreme thermophilie: 60 – 80oC
Hyperthermophile: 80 and above
A new bug (strain
121) grow in
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121) grow in
85~121oC (Kashefi,
2003)
Unsaturated
fatty acid
membraneSaturated fatty acid membrane
Psychrophile in cold environment
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Snow algae
Spore of
Chlamydomonas
nivalis
Psychrophile in cold environment
Molecular adaptation to psychrophily
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Molecular adaptation to psychrophily
• Cold-active enzymes have more α-helix and lesser ß-sheet secondary structure
→greater flexibility in the cold
• Cell membrane of psychrophiles has higher content of
unsaturated fatty acids
→maintain membrane fluidity
Growth of hyperthermophiles in boiling water
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Molecular adaptation to thermophily
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Molecular adaptation to thermophily
• Thermostable enzymes differ very little in a.a. sequence
from heat-labile enzymes, but critical a.a. substitution insome locations.
• Increased ionic bonds b/w a.a. residues
→densely packed highly hydrophobic interiors of proteins
→resist unfolding of proteins in aqueous cytoplasm• Produce high levels of solutes in cytoplasm to stabilize
proteins against thermal degradation.
• Cell membrane of thermophiles has higher content of
saturated fatty acids to form a stronger hydrophobic state→maintain membrane stability
Temperature effect on growth
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Arrhenius equation
µ =A*e(-Ea/R*T)
µ : microbial growth rate
A: frequency factor, a constant (frequency of collisions and their orientation); varies slightly withtemperature, but constant across small temperature
ranges
Ea: activation energy (minimum energy needed for the
reaction to occur, J mol-1 )
R: universal gas constant (8.314 J mol-1K-1)
T: absolute temperature (oK)
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Picrophilus
oshimae, pHopt
~0.7
Bacillus firmus,pH range 7~11
Osmotic effect on growth
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Os ot c e ect o g owt
water availability
usually expressed water activity (a
w)
aw: ratio of vapor
pressure of air inequilibrium with a
solution to the vaporpressure of pure water;vary b/w 0~1.
H2O high water concentration
low water concentration
Water activity (aw
) Material
1.000 Pure water
0.995 Human blood
0.980 Seawater
0.900 Maple syrup
0.800 Jams
0.750 Salt lakes
0.700 Cereals, dried
fruit
Osmosis
Water activity (Osmolarity)
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In natural habitats, osmotic effects mainlywith high concentration of salts.
Halophile: require NaCl for growth
based on optimal NaCl concentration([NaCl] of seawater: 3%)
mild halophile: 1 - 6%
moderate halophile: 6 - 15%
extreme halophile: 15 – 30%
Osmophile: live in high-sugar environment
Xerophile: live in very dry environment
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Molecular adaptation to low water activity
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p y
• Increasing cell internal solute concentration by
1. pumping inorganic ions (K+) into cell from theenvironment
2. synthesize and accumulate an organic solute
(Compatible solutes, 相容溶質 )
• Compatible solutes are highly water-soluble sugar,alcohols, amino acids, and their derivatives
Common organic
compatible solutes
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compatible solutes
in halophiles
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Classes of microbes based on oxygen level
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a. Obligate(Strict) aerobes: atmospheric O2 concentration
(21%); respire O2 during metabolismb. Obligate(Strict) anaerobes: no O2 at all, growth
inhibited or killed if it presents
c. Facultative aerobes: aerobes not require O2
for growth,
but do better in its presence
d. Microaerophiles: aerobes need only 2 –10% O2;
damaged by atmospheric O2 concentration
e. Aerotolerant anaerobes: not require O2 for growth and
grow better in its absence; O2 was not used.
Growth in
tubes of
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thioglycolate
broth
Oxic zone
Anoxic zone
GasPak
Anaerobic
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Anaerobic
Jar
2H 2 + O2 → 2H 2O on
Pd
Headspace: N2, H2, CO2
rubber gasket
air-tight seal
Add 10 ml H2O to
generate H2 & CO2
Sodium borohydride,
sodium bicarbonate,
citric acid, palladium
Growth of methanogen a strict anaerobe
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Growth of methanogen, a strict anaerobe
• Not only to remove traces of O2 but also carry out allmanipulations of cultures in an anoxic atmosphere.
• Hungate technique
Boiled medium and then reducing agent (H2S or Na2S) is
added.
All manipulations are carried out under a jet of O2-free
H2 or N2 gas that is blown into open culture vessel,
driving out any oxygen that might enter.
Work with open cultures can be done in anoxic glove
box in completely anoxic atmosphere.
Gas station
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過氧化酶
超氧化物歧化酶
超氧化物還原酶 , in certain anaerobes