Atmospheric

Habitat
 Gain a basic understanding of
atmospheric microbes

Explain the diversity, occurrence and

distribution of microbes in atmosphere
 Composition

79% Nitrogen (N2)

21% Oxygen (O2)
0.034% Carbon Dioxide (CO2)
trace Other gases
Regions in atmosphere
a) Troposphere
- Nearest to Earth
- Composition of air??
- Characteristics:
Air temperature
atm pressure Decrease with
conc. O2 altitude
organic C
water
radiation Increase with altitude
- Most highly populated area among regions
 Troposphere - from surface to about 10 km

temperature at upper levels reach -43 to

-83C

upper levels too extreme to support life

location of the bad ozone

b) Stratosphere
- Increasing temperature with height
-10 km to 45 km

location of good ozone, limits UV light

extreme low temperatures

lacking liquid water

c) Mesosphere
- Temperature decreasing with height

d) Thermosphere
- Temperature increases with altitude

e) Exosphere
- Extending into outer space
- Helium and H2 predominant
 Ionosphere 45 km to 100 km

extreme low temperatures

lacking liquid water

extreme UV and ionizing radiation levels

 The atmospheric habitat

the lower troposphere contains high numbers of

microbes

thermal gradients cause rapid mixing, allowing for

dispersal of microbes

air movement provides means of dispersal

precipitation aids deposition
radiation, temperature, humidity affect survival
 The atmospheric habitat - Dispersal vs. Growth

The lower atmosphere can theoretically support growth

clouds have sufficient liquid water

light intensity within appropriate limits
condensation nuclei could act as substrata for
photoautotrophs
some locations (industrial areas) have
sufficient organic content to support
heterotrophic growth
- Spores are better adapted to atmosphere
because

a) they have low metabolic rates

b) are produced in high numbers
c) thick cell wall against desiccation
d) some are pigmented protection against UV
radiation
e) small size and low density airborne
 Most microbes in the atmosphere are
in some resting or resistant form
xenospores - forms that are primarily adapted to
reproduction vs. resistance to environmental
stress
fungi
algae
protozoans
bacteria (e.g. actinomycetes)
endospores - forms that are primarily adapted to
resistance rather than reproduction

limited to Gram-positive bacteria

 xenospores vs. endospores

xenospores form on or in specialized structures

endospores form within the vegetative cell

xenospores represent an adaptation for fecundity

one cell many spores survival rate low

endospores represent an adaptation for survival

one cell one spore survival rate high
 xenospores and endospores
both are more resistant than parental cells
very low metabolic rates (low maintenance
energy)
thick cell walls protect against desiccation

pigmentation protects against UV light and

photooxidation
many have high surface area:weight ratios
 survival in an air-dried state
2 - 6 minutes for Vibrio cholerae
~ 106 years for coryneforms, Gram-positive
cocci
> 25 million years for some Bacillus spores

biomass in the atmosphere (surface to 3 km)

101 to 104 cells per cm3
Spores or resting stages can enter the
atmosphere dry or in aerosols (rain-splash
or sneezes) and travel great distances.

During a sneeze, millions of tiny

droplets of water and mucus are
expelled at about 200 miles per
hour (100 metres per second). The
droplets initially are about 10-100
m in diameter, but they dry rapidly
to droplet nuclei of 1-4 m,
containing virus particles or
bacteria. This is a major means of
transmission of several diseases of
humans.
- Favorable conditions for dispersal with

a) higher wind speed

b) lower humidity
- Dispersal of spores and vegetative m/os through
a) passive mechanism with dust particles, aerosols.
b) active mechanism e.g. bursting of sporangium

- M/os - removed from the atmosphere by

a) gravitational forces
b) rain
c) precipitation e.g. dust particles
Pseudomonas syringae is probably the most abundant ice
nucleating bacteria in air, rain and hail (Sands et al. 1982)
 Pseudomonas syringae is a common foliar pathogen

Pseudomonas syringae was ALSO the subject of early

biotechnology research because of its ice nucleating properties
Physical features of the aerial environment
a) Air movement in the troposphere
- Main agent of dispersal
- Kinetic energy for movement of air provided by
solar energy
- Principle:
Temperature decreases with altitude
Sun heats up the ground, create gradient
Displacement of air occur
b) Radiation
- Radiation from the sun
- Mostly absorbed or reflected back but m/os
at higher region of troposphere may be
exposed to damaging doses
c) Precipitation
- Water vapor continually recycled.
- Average time of water vapor in air = about 9 days
Dispersal of airborne particles
- 3 stages
a.) liberation and take-off into the air
b.) dispersion in air currents
c.) deposition
Liberation
- Requires energy to overcome adhesive forces and
still air layer
- Features of adaptation for airborne dispersal:
virus and bacteria are poorly adapted
fungi have many adaptation features
e.g.
a.) tall sporophores
b.) forceful ways to project spores into air
Dispersion
- Depend on physical characteristics of particle (e.g.
spore) and the atmosphere
- Spore:
size
shape
degree of surface roughness
density
- Environment:
wind movement
turbulence
air humidity
convection (changes of air due to temperature)
Deposition
- Methods
a.) Sedimentation due to gravity
b.) Impaction with surfaces
c.) Brownian movement
d.) Electrostatic deposition
e.) Rainwashing
Other extreme environments
-With severe conditions
-high or low temperature
- pH
-irradiation
-toxic chemicals
- lack of nutrients, water availability
- ionizing radiation.
Hot Springs
- At volcanic areas
- Grow of m/os limited by
a.) high temperature
b.) high acidity or alkalinity
c.) low concentration of organic matter
d.) low concentration of dissolved oxygen
- Predominant bacterium: Bacillus stearothermophilus

Acid springs and lakes

- pH 3 or less
 Ferroplasma acidarmanus
pH 0.0
Capable of massive surface growth in flowing
waters in the subsurface
Possesses a single peripheral cytoplasmic
membrane
No cell wall
Salt lakes
- High concentration of sodium chloride
- High salt causes
a.) dehydration
b.) precipitation of intracellular proteins
- Predominant bacteria: Halobacterium
- Adaptation feature:
a) Cell structure and enzyme naturally require
stabilization from sodium
-Require a sodium ion of min [1.5M], optimum [3-4
M]
Antarctica
- Environmental condition
low temperature
low humidity
high velocity of desiccating winds
high radiation
- Community diversity is low
- Food web 3-4 levels of species
The Antarctica Marine Food Web
Carnivores e.g. penguins, seals,
seabirds, fish and squid

Krill is the key or

central species of
the marine food web

Photosynthetic organisms e.g.

phytoplankton
 Stress Environmental Microorganisms observed
Conditions

High T 121oC Geogemma barossii

110-113 oC, deep Pyrolobus fumarii
marine trenches Methanopyrus kandleri
Pyrodictium abyssi

67-102 oC, marine Pyrococcus abyssi

basins

85 oC, hot springs Thermus sulfolobus

75 oC, sulfur hot Thermothrix thiopara
springs

Low T -12 oC, Antarctic ice Psychromonas ingrahamii

Osmotic stress 13-15% NaCl Chalmydomonas

25% NaCl Halobacterium,Halococcus
 Stress Environmental Microorganisms observed
Conditions

pH pH 10 or above Bacillus

pH 3 or lower Sachharomyces
Thiobacillus
pH 0.5 Picrophilus oshimae

pH0.0 Ferriokasna acidarmanus

Low water Aw = 0.6-0.65 Torulopsis

availability Candida

T and low pH 85 oC, pH1.0 Cyanidium

Sulfolobus acidocaldarum

Pressure 500-10.5 atm Colwellia hadaliensis

Radiation 1.5 million rads Deinococcus radiodurans

 Microbial ecology methods:
Microscopic
Cultural
Physical
Chemical
Molecular techniques
Measurement of microbial numbers
and activities
- Three phases of determination:
a) Sample collection
b) Sample processing
c) Actual measurement
Sample collection
- Different approach
- Method used is determined by
a.) Chemical and physical properties of ecosystem
b.) Expected abundance of m/os
c.) Enumeration or measurement procedures to be
performed
- Precautions during sampling
a.) Numbers or activities of m/os are not altered
b.) Samples are not contaminated
Soil samples
- Not necessary to use aseptic technique
- Surface soil use shovel
Particular depth use soil corer
- Sampling of soil m/os present in low number:
by absorption and concentration of naturally
occurring dissolved nutrients e.g. Cholodny-Rossi
buried slide technique

Variations in technique:
- Use electron microscope grids
- Use flattened glass capillaries (pedoscopes soil;
peloscopes sediments).
Water samples
- Devices:
a.) to collect water from known depths
b.) withstand decompression problems
c.) control to open and close for sample collection
Air samples
Sampling air for m/os

- Principles of techniques
sedimentation e.g. settle plate method
filtration

Two approaches
a. Active
b. Passive
Active approach:

- Known volume of air aspirated

- M/o collected on agar surface
- Unit= cfu/m3
Passive approach:
- Area defined
- M/o settle by gravity on agar plates
- Unit = cfu/m2/h
Advantages of passive method:
a.) convenient
b.) cheap

Disadvantages of passive method:

a.) cannot be related to volume of air sampled
b.) rate of deposition varies with terminal
velocity
Filtration Method
- Using membrane, glass fiber or alginate filters
- Limitation: excessive desiccation
- Filters may collect cells through sieving,
impaction, electrostatic attraction, alone or
combination
- M/os from membrane filters can be obtained for
further studies
Sample processing
- Diluted or concentrated
- Precaution: Bottle effect
- When dilution is required, recovery is dependent on
chemical composition and osmotic strength of
the diluent
time of mixing
temperature
degree of agitation
**varies from sample to sample **
- If concentration by filtration
pore size
flatness and uniformity of the pores
chemical composition of the filter
- Processing condition compatible
- Enumeration procedure is not to discriminate
between living and dead m/os samples can be
preserved
Actual measurement
Different methods depending on what you
want to study
Examination of Microbial Communities as
Complex Assemblages
- Difficulties due to complexity
- E.g. bulk DNA extraction
Problems:
1. DNA obtained vary with method
2. DNA recovered may be from non living
organisms
3. DNA recovered from nonfunctional
organisms
Different methods directed toward
different community features such as:
a) Numbers and types of microbes
b) Microbial community structure and constituents
c) Microbial viability and stress sensitivity
d) Microbial activity at various time scales and
levels of turnover
a) Numbers and Types of Microbes
- Various techniques
- Strategy: Duplicate the resources and
conditions in the niche
- Use enrichment medium, selective medium
1 Streak plate

2 Agar shake tube method

- Variations: Specific agar medium and can
be enumerated
3 Liquid dilution method:
- Dilution to extinction using liquid
medium
- Variations: specific medium and
enumeration
Series of dilution
10-1, 10-2, 10-3, 10-4.
*

incubation

A B C D A B C D
Single colony Check purity
1. Microscopy
2. Growth in medium
that favors growth
of contaminants
Enrichment bias
- Did not enrich the dominant members of the
community due to unexplained reasons
- Overcome by extinction dilution method
dilute out the others.
b) Microbial community structure and
constituents
- e.g. direct microscopy observation or coupled
with molecular probe
Staining techniques
- Can be used for enumeration
- Methods:
a. Fluorescent staining
b. Viability staining
c. Fluorescent antibodies
Fluorescent Staining:
- Fluorescent dyes: Acridine orange
DAPI

Bind to nucleic
Bind to nucleic acid.
acid.
Fluoresce bright blue
Fluorescent
microscope
green or orange
Advantages of Fluorescent staining:
a. Non-specific: stain all m/os in sample
b. Easy to see and enumerate (total)

Disadvantages of Fluorescent staining:

a. Do not differentiate living/dead
b. Do not allow tracking specific m/os
Viability Staining:
- Green fluorescent dye
Red fluorescent dye
Penetrate
living/dead cells

Penetrate cells without

intact cell membrane
dead
Advantage of viability staining:
a. No. of viable m/os in sample

Disadvantage of viability staining:

a. Nonspecific staining of background
materials
Fluorescent Antibodies:
- Antibodies prepared against surface
constituents of particular organism
- Label with fluorescent
Advantage of viability staining:
a. Highly specific suitable for
identification and tracking of m/os

Disadvantage of viability staining:

a. Time consuming and laborious to
prepare antibodies
Genetic Stains and Community Analysis
- Nucleic acid probe (DNA/RNA)
labelled with fluorescent dye
- Attach to complementary sequence
fluoresce
- Fluorescent in situ hybridization (FISH)
- cytogenetic technique that can be used to detect
and localize the presence or absence of specific
DNA sequences on chromosomes
Applications of FISH technologies:
a. Phylogenetic staining
- Signature sequence
- Different sequence study interaction of
m/os
c) Stress and viability of m/os
- Stress e.g. absence of nutrients, toxic
chemicals, temperature changes
- Can be measured by cultural methods
d) Microbial activity and turnover
- Direct chemical measurement (HPLC,
GC)
- Stable isotope measurement,
radioisotopes and microelectrodes
Radioisotopes
- Measurement of collective activities by several
metabolically related populations by measuring
single transformation (substrate product)
-E.g. Photoautotrophic activity measure
uptake of 14CO2 into microbial cells
Chemoorganotrophic activity measure
incorporation of 14C-organic compounds into
bacterial biomass
Caution:
- Transformation due to chemical processes
- Killed cell control to eliminate background
Microelectrodes
- Small glass electrode to measure pH,
oxygen, CO2, H2 or H2S and etc. in
microenvironment
 Stable isotopes

- Stable isotope measurements indicate whether C, N,

or other elements have been processed by the
microbes.
-Carbon: 12C/13C
Sulfur: 32S, 34S
- Most biochemical reactions favor lighter isotope
(12C, 32S)
-When a microbe uses CO2 or an organic substrate,
cells and their metabolic products lower
concentrations of the heavy isotope than does the
original substrate.
I know quite certainly that I myself have no special
talent; Curiosity, obsession and dogged endurance,
combined with self-criticism, have brought me to
my ideas
-Albert Einstein