chapter 5 - marmara Üniversitesimimoza.marmara.edu.tr/~deniz.tufan/enve3003/lecture5.pdflecture...

LECTURE PRESENTATIONS

For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION

Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark

Lectures by

John Zamora

Middle Tennessee State University

© 2012 Pearson Education, Inc.

Microbial Growth

Chapter 5

I. Bacterial Cell Division

• 5.1 Cell Growth and Binary Fission

© 2012 Pearson Education, Inc. Marmara University – Enve3003 Env. Eng. Microbiology – Assist. Prof. Deniz AKGÜL

5.1 Cell Growth and Binary Fission

• Growth: increase in the number of cells

• Binary fission: cell division following enlargement

of a cell to twice its minimum size (Figure 5.1)

• Generation time: time required for microbial cells

to double in number

• During cell division, each daughter cell receives a

chromosome and sufficient copies of all other cell

constituents to exist as an independent cell


Animation: Binary Fission

Animation: Overview of Bacterial Growth

Marmara University – Enve3003 Env. Eng. Microbiology – Assist. Prof. Deniz AKGÜL

binary_fission.html

bacterial_growth.html

Figure 5.1 Binary fission in a rod-shaped prokaryote


Septum

Cellelongation

Septumformation

Completionof septum;formation ofwalls; cellseparation

On

e g

en

era

tio

n


II. Population Growth

• 5.5 The Concept of Exponential Growth

• 5.6 The Mathematics of Exponential Growth

• 5.7 The Microbial Growth Cycle

• 5.8 Continuous Culture: The Chemostat


5.5 The Concept of Exponential Growth

• Most bacteria have shorter generation times

than eukaryotic microbes

• Generation time is dependent on growth

medium and incubation conditions


5.5 The Concept of Exponential Growth

• 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 (Figure 5.8)


Figure 5.8 The rate of growth of a microbial culture


Time (h)

Logarithmicplot

Arithmeticplot

Nu

mb

er

of c

ell

s(a

rith

me

tic

sc

ale

)

Nu

mb

er

of c

ell

s(l

og

ari

thm

ic s

cale

)1000

500

100

0 1 2 3 4 5 1

10

102

103


5.6 The Mathematics of Exponential

Growth

• A relationship exists between the initial number of

cells present in a culture and the number present

after a period of exponential growth:

N = N02n

N is the final cell number

N0 is the initial cell number

n is the number of generations during the

period of exponential growth



Growth

• Generation time (g) of the exponentially

growing population is

g = t/n

t is the duration of exponential growth

n is the number of generations during

the period of exponential growth



Growth

• Specific growth rate (k) is

calculated as

k = 0.30/g

• Division rate (v) is

calculated as

v = 1/g


Figure 5.9 Calculating microbial growth parameters.

Method of estimating the generation times (g) of

exponentially growing populations with generation

times of 2 h from data plotted on semilogarithmic

graphs. The slope of line is equal to 0.30/g, and n is

the number of generations in the time t.

0.152

log(2)Slopek


5.7 The Microbial Growth Cycle

• Batch culture: a closed-system microbial culture

of fixed volume

• Typical growth curve for population of cells grown

in a closed system is characterized by four

phases (Figure 5.10):

– Lag phase

– Exponential phase

– Stationary phase

– Death phase


Animation: Bacterial Growth Curve


bacterial_growth_curve.html

Figure 5.10 Typical growth curve for a bacterial population

Time

Lo

g10 v

iab

le

org

an

ism

s/m

l

Op

tical

den

sit

y (

OD

) 10

9

8

7

6

1.0

0.75

0.50

0.25

0.1

Viable count

Turbidity (optical density)

Lag Exponential Stationary Death

Growth phases


A viable count measures the cells in the culture that are capable of

reproducing. Optical density (turbidity), a quantitative measure of light

scattering by a liquid culture, increases with the increase in cell number.



• Lag phase

– Interval between when a culture is inoculated

and when growth begins

• Exponential phase

– Cells in this phase are typically in the

healthiest state

• Stationary phase

– Growth rate of population is zero

– Either an essential nutrient is used up or

waste product of the organism accumulates

in the medium



• Death Phase

– If incubation continues after cells reach

stationary phase, the cells will eventually die


5.8 Continuous Culture: The Chemostat

• Continuous culture: an open-system microbial

culture of fixed volume

• Chemostat: most common type of continuous

culture device (Figure 5.11)

– 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 is pumped out (mean cell

residence time or hydraulic retention time ‘HRT’)

• Concentration of a limiting nutrient


Figure 5.11 Schematic for a continuous culture device (chemostat)


Fresh mediumfrom reservoir

Sterile air orother gas

Flow-rateregulator

Gaseous headspace

Culturevessel

Culture

Overflow

Effluent containingmicrobial cells

The population density is controlled by the concentration of limiting

nutrient in the reservoir, and the growth rate is controlled by the flow rate.

Both parameters can be set by the experimenter. Marmara University – Enve3003 Env. Eng. Microbiology – Assist. Prof. Deniz AKGÜL


• 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 (Figure 5.12)


Figure 5.12 The effect of nutrients on growth


Only yield affectedG

row

th r

ate

( )

Nutrient concentration (mg/ml)

Gro

wth

yie

ld (

)

0 0.1 0.2 0.3 0.4 0.5

Rate andyield affected

Relationship between nutrient concentration, growth rate (green curve),

and growth yield (red curve) in a batch culture (closed system). Only at

low nutrient concentrations are both growth rate and growth yield affected.



• Chemostat cultures are sensitive to the dilution

rate and limiting nutrient concentration

(Figure 5.13)

– At too high a dilution rate, the organism is

washed out

– At too low a dilution rate, the cells may die from

starvation

– Increasing concentration of a limiting nutrient

results in greater biomass but same growth

rate


Figure 5.13 Steady-state relationships in the chemostat


5 Bacterial concentration

Do

ub

lin

g t

ime (

h)

Ste

ad

y-s

tate

bacte

rial

co

ncen

trati

on

(g

/l)

Steady state

Washout

4

3

2

1

00 0.25 0.5 0.75 1.0

4

6

2

0

Dilution rate (h1)

At high dilution rates, growth cannot balance dilution, and the population

washes out. Although the population density remains constant during

steady state, the growth rate (doubling time) can vary over a wide range.


III. Measuring Microbial Growth

• 5.9 Microscopic Counts

• 5.10 Viable Counts

• 5.11 Turbidimetric Methods


5.9 Microscopic Counts

• Microbial cells are enumerated by microscopic

observations (Figure 5.14)

– Results can be unreliable


Figure 5.14 Direct microscopic counting procedure using the Petroff–Hausser counting chamber



• Limitations of microscopic counts

– Cannot distinguish between live and dead cells

without special stains

– Small cells can be overlooked

– Precision is difficult to achieve

– Phase-contrast microscope required if a stain is

not used

– 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

– Uses laser beams, fluorescent dyes, and

electronics


5.10 Viable Counts

• Viable cell counts (plate counts):

measurement of living, reproducing

population

– Two main ways to perform plate counts:

• Spread-plate method (Figure 5.15)

• Pour-plate method

• To obtain the appropriate colony number, the

sample to be counted should always be

diluted (Figure 5.16)


Figure 5.15 Two methods for the viable count

Sample is pipetted onto

surface of agar plate

(0.1 ml or less)

Sample is spread evenly

over surface of agar

using sterile glass

spreader

Typical spread-plate

results

Surface

colonies

Surface

colonies

Subsurface

colonies

Typical pour-plate

results Sample is pipetted into

sterile plate

Sterile medium is

added and mixed

well with inoculum

Spread-plate method

Pour-plate method

Incubation

Solidification

and incubation


Colonies of

Escherichia coli

formed from cells

plated by the spread-

plate method (top) or

the pour-plate

method (bottom)

Colonies form within

the agar as well as

on the agar surface


Figure 5.16 Procedure for viable counting using serial dilutions of the sample and the pour-plate method


Sample to

be counted1 ml

1 ml 1 ml 1 ml 1 ml 1 ml

9-ml

broth

Total dilution

Plate 1-ml samples

1/10

(101)

1/100

(102)

1/103

(103)

1/104

(104)

1/105

(105)1/106

(106)

159 103 1.59 105Plate

countDilution

factor

Cells (colony-forming units)

per milliliter of original sample

159

colonies

17

colonies

2

colonies0

coloniesToo many colonies

to count

The sterile liquid used for

making dilutions can simply

be water, but a solution of

mineral salts or actual

growth medium may yield a

higher recovery.


5.10 Viable Counts

• Plate counts can be highly unreliable when

used to assess total cell numbers of natural

samples (e.g., soil and water)

– Selective culture media and growth conditions

target only particular species


5.10 Viable Cell Counting

• The Great Plate Anomaly: direct microscopic

counts of natural samples reveal far more

organisms than those recoverable on plates

• Why is this?

– Microscopic methods count dead cells

whereas viable methods do not

– Different organisms may have vastly different

requirements for growth


5.11 Turbidimetric Methods

• Turbidity measurements are an indirect, rapid,

and useful method of measuring microbial

growth (Figure 5.17a)

– Most often measured with a spectrophotometer

and measurement referred to as optical density

(O.D.)


Figure 5.17a Turbidity measurements of microbial growth


Light

Prism

Incident light, I0

Filter

Sample containing

cells ( )

Unscattered light, I

Photocell (measures

unscattered light, I )

Spectrophotometer

Optical density (OD)

LogI0

I

Measurements of turbidity

are made in a

spectrophotometer.

The photocell measures

incident light unscattered by

cells in suspension and

gives readings in optical

density units.



• To relate a direct cell count to a turbidity

value, a standard curve must first be

established (Figure 5.17c)


Figure 5.17c Turbidity measurements of microbial growth

Theoretical

Actual

Cell numbers or mass

(dry weight)

Op

tical

den

sit

y

0.8

0.6

0.4

0.3

0.2

0.1

0.5

0.7


The one-to one

correspondence between

these relationships breaks

down at high turbidities

Relationship between cell

number or dry weight and

turbidity readings.



• 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)


IV. Temperature and Microbial

Growth

• 5.12 Effect of Temperature on Growth

• 5.13 Microbial Life in the Cold

• 5.14 Microbial Life at High Temperatures


5.12 Effect of Temperature on Growth

• Temperature is a major environmental factor

controlling microbial growth

• Cardinal temperatures: the minimum,

optimum, and maximum temperatures at

which an organism grows (Figure 5.18)


Figure 5.18 The cardinal temperatures: minimum, optimum, and maximum


Enzymatic reactions occurringat maximal possible rate

Enzymatic reactions occurringat increasingly rapid rates

Membrane gelling; transportprocesses so slow that growthcannot occur

Protein denaturation; collapseof the cytoplasmic membrane;thermal lysis

Temperature

Gro

wth

ra

te

Minimum

Optimum

Maximum

Actual

values

may vary

greatly for

different

organisms



• Microorganisms can be classified into groups by

their growth temperature optima (Figure 5.19)

– Psychrophile: low temperature

– Mesophile: midrange temperature

– Thermophile: high temperature

– Hyperthermophile: very high temperature


Figure 5.19 Temperature and growth response in different temperature classes of microorganisms


Temperature (oC)

Gro

wth

ra

te

Psychrophile

Mesophile

Thermophile

Hyperthermophile Hyperthermophile

0 10 20 30 40 50 60 70 80 90 100 110 120

4°

39°

60°88° 106°

Example:Polaromonas vacuolata

Example:Escherichia coli

Example:Geobacillusstearothermophilus Example:

Thermococcus celer

Example:Pyrolobus fumarii



• Mesophiles: organisms that have midrange

temperature optima; found in

– Warm-blooded animals

– Terrestrial and aquatic environments

– Temperate and tropical latitudes


5.13 Microbial Life in the Cold

• Extremophiles

– Organisms that grow under very hot or very cold conditions

• Psychrophiles

– Organisms with cold temperature optima

– Inhabit permanently cold environments (Figure 5.20)

• Psychrotolerant

– Organisms that can grow at 0ºC but have optima of 20ºC to 40ºC

– More widely distributed in nature than psychrophiles



• Molecular Adaptations to Psychrophily

– Production of enzymes that function optimally

in the cold; features that may provide more

flexibility

• More -helices than -sheets

• More polar and less hydrophobic amino acids

• Fewer weak bonds

• Decreased interactions between protein

domains



• Molecular Adaptations to Psychrophily (cont’d)

– Transport processes function optimally at low

temperatures

• Modified cytoplasmic membranes

– High unsaturated fatty acid content


5.14 Microbial Life at High Temperatures

• Above ~65 oC, only prokaryotic life forms exist

• Thermophiles: organisms with growth temperature

optima between 45 oC and 80 oC

• Hyperthermophiles: organisms with optima greater

than 80 oC

– Inhabit hot environments including boiling hot

springs and seafloor hydrothermal vents that can

have temperatures in excess of 100 oC



• Hyperthermophiles in Hot Springs

– Chemoorganotrophic and chemolithotrophic

species present (Figure 5.22)

– High prokaryotic diversity (both Archaea and

Bacteria represented)


Figure 5.22 Growth of hyperthermophiles in boiling water


(a) Boulder Spring, a

small boiling spring in

Yellowstone National

Park. This spring is

superheated, having a

temperature 1-2 oC above

the boiling point. The

mineral deposits around

the spring consist mainly

of silica and sulfur.

(b) Photomicrograph of a

microcolony of

prokaryotes that

developed on a

microscope slide

immersed in such a

boiling spring.



• 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



• 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,

diglycerol phosphate) help stabilize proteins



• Molecular Adaptations to Thermophily (cont’d)

– Modifications in cytoplasmic membranes to

ensure heat stability

• Bacteria have lipids rich in saturated fatty acids

• Archaea have lipid monolayer rather than bilayer


V. Other Environmental Factors

Affecting Growth

• 5.15 Acidity and Alkalinity

• 5.16 Osmotic Effects on Microbial Growth

• 5.17 Oxygen and Microorganisms

• 5.18 Toxic Forms of Oxygen


5.15 Acidity and Alkalinity

• The pH of an environment greatly affects

microbial growth (Figure 5.24)

• Some organisms have evolved to grow best

at low or high pH, but most organisms grow

best between pH 6 and 8 (neutrophiles)


Figure 5.24 The pH scale


Volcanic soils, watersGastric fluidsLemon juiceAcid mine drainageVinegar

RhubarbPeaches

Acid soilTomatoes

American cheeseCabbage

PeasCorn, salmon, shrimp

Pure water

Seawater

Very alkalinenatural soil

Alkaline lakes

Soap solutions

Household ammoniaExtremely alkaline

soda lakes

Lime (saturated solution)

Neutrality

Increasingacidity

Increasingalkalinity

107 107

Ac

ido

ph

ile

sA

lka

lip

hile

s

pH Example Moles per liter of:H OH

101

102

103

104

105

106

108

109

1010

1011

1012

1013

1014

1014

1013

1012

1011

1010

109

108

106

105

104

103

102

101

1

1

Although some

microorganisms

can live at very low

or very high pH,

the cell’s internal

pH remains near

neutrality.



• Acidophiles: organisms that grow best at low

pH (<6)

– Some are 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



• 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 as high as 9.5 in extreme acido- and

alkaliphiles, respectively

• Microbial culture media typically contain

buffers to maintain constant pH


5.16 Osmotic Effects on Microbial Growth

• Water activity (aw): water availability;

expressed in physical terms

– Defined as ratio of vapor pressure of air in

equilibrium with a substance or solution to the

vapor pressure of pure water



• Typically, the cytoplasm has a higher solute

concentration than the surrounding

environment, thus the tendency is for water to

move into the cell (positive water balance)

• When a cell is in an environment with a higher

external solute concentration, water will flow

out unless the cell has a mechanism to

prevent this



• Halophiles: organisms that grow best at reduced

water potential; have a specific requirement for

NaCl (Figure 5.25)

• Extreme halophiles: organisms that require high

levels (15–30%) of NaCl for growth

• Halotolerant: organisms that can tolerate some

reduction in water activity of environment but

generally grow best in the absence of the added

solute


Figure 5.25 Effect of sodium chloride (NaCl) concentration on growth of microorganisms of different salt

tolerances or requirements.


Example:Staphylococcusaureus

Example:Aliivibrio fischeri Example:

Halobacteriumsalinarum

Example:Escherichia coli

NaCl (%)

Gro

wth

ra

te

0 5 10 15 20

Nonhalophile

Halotolerant Halophile Extremehalophile

Optimum NaCl concentration for extreme halophiles, it is between 15-30%



• Osmophiles: organisms that live in

environments high in sugar as solute

• Xerophiles: organisms able to grow in very

dry environments



• Mechanisms for combating low water activity in

surrounding environment involve increasing the

internal solute concentration by

– Pumping inorganic ions from environment

into cell

– Synthesis or concentration of organic solutes

• compatible solutes: compounds used by cell to

counteract low water activity in surrounding

environment


5.17 Oxygen and Microorganisms

• 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



• Thioglycolate broth (Figure 5.26)

– Complex medium that separates microbes

based on oxygen requirements

– Reacts with oxygen so oxygen can only

penetrate the top of the tube


Figure 5.26 Growth versus oxygen (O2) concentration


Oxic zone

Anoxic zone

(a) O2 penetrates only a short

distance into the tube, so

obligate aerobes grow only

close to the surface.

(b) Anaerobes, being sensitive to

O2, grow only away from the

surface.

(c) Facultative aerobes are able to

grow in either the presence or

the absence of O2 and thus

grow throughout the tube.

However, growth is better near

the surface because these

organisms can respire.

(d) Microaerophiles grow away

from the most oxic zone.

(e) Aerotolerant anaerobes grow

throughout the tube. Growth is

not better near the surface

because these organisms can

only ferment.

The redox dye, resazurin, which is pink when oxidized and colorless when reduced, has

been added as a redox indicator.

Aero

bes

Anaero

bes

Facultative a

ero

bes

Mic

roaero

phile

s

Aero

tole

rant

anaero

bes



• Special techniques are needed to grow

aerobic and anaerobic microorganisms

(Figure 5.27)

• Reducing agents: chemicals that may be

added to culture media to reduce oxygen

(e.g., thioglycolate)


Figure 5.27 Incubation under anoxic conditions


A chemical reaction in the envelope

in the jar generates H2 + CO2. The H2

reacts with O2 in the jar on the

surface of a palladium catalyst to

yield H2O; the final atmosphere

contains N2, H2, and CO2.

Anoxic jar

Anoxic glove bag for manipulating and incubating cultures under anoxic

conditions. The airlock on the right, which can be evacuated and filled

with O2-free gas, serves as a port for adding and removing materials to

and from the glove bag.

Anoxic glove bag


5.18 Toxic Forms of Oxygen

• Several toxic forms of oxygen can be formed

in the cell (Figure 5.28):

– Single oxygen

– Superoxide anion

– Hydrogen peroxide

– Hydroxyl radical


Figure 5.28 Four-electron reduction of O2 to H2O by stepwise addition of electrons


All intermediates formed are reactive and toxic to cells, except for water

Reactants Products

Outcome:

(superoxide)

(hydrogen peroxide)

(hydroxyl radical)

(water)


5.18 Toxic Forms of Oxygen

• Enzymes are present to neutralize most of

these toxic oxygen species

– Catalase

– Peroxidase

– Superoxide dismutase

– Superoxide reductase


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