Class 5 Growth

8/14/2019 Class 5 Growth

http://slidepdf.com/reader/full/class-5-growth 1/68

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)



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



Microbial Growth

Yeast is an example of

microbe that reproduces by budding





All cellcomponents

double.



• 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





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.



• 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.



Growth Curve (Batch culture)



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



Exponential

growth



Growth Parameters



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



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



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=



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



- 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



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



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



@ 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





µ =µ max ⇒zero

order

µ ∝ [S]⇒first

order





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



Chemostat





Steady-state relationships in the chemostat



Steady-state relationships in the chemostat

Measurement of growth



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)



Direct microscopic counting



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



Viable count (I)

Vi bl



Viable

count (II)







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)











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.





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



121) grow in

85~121oC (Kashefi,

2003)

Unsaturated

fatty acid

membraneSaturated fatty acid membrane

Psychrophile in cold environment



Snow algae

Spore of

Chlamydomonas

nivalis

Psychrophile in cold environment

Molecular adaptation to psychrophily



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



Molecular adaptation to thermophily



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



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)



Picrophilus



Picrophilus

oshimae, pHopt

~0.7

Bacillus firmus,pH range 7~11

Osmotic effect on growth



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)



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



Molecular adaptation to low water activity



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



compatible solutes

in halophiles





Classes of microbes based on oxygen level



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



thioglycolate

broth

Oxic zone

Anoxic zone

GasPak

Anaerobic



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



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



Anoxic glove box



Oxygen toxicity



觸酶、過氧化氫酶



過氧化酶

超氧化物歧化酶

超氧化物還原酶 , in certain anaerobes



Class 5 Growth

Documents