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Microbial ecology
VL 1
Microbial abundance
and ecological range
Bert Engelen [email protected]
www.icbm.de/pmbio
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Bacteria are quite small..........
Bacterium app. 1 µm
For a bacterium, one cm3 appears to be like acube of 20 km for us!
x 2 · 106
Men app. 2 m
x 2 · 106
4000 km (Gibraltar – Northern cape)
..........but there are lots of them!
- Intestines 0.05 · 1029
- Oceans, app. 106 ml-1 1029
- Soil 2.6 · 1029
- Limnic systems 0.002 · 1029
- Sediments 0.2 · 1029
- Subsurface 40-60 · 1029
Whitman et al., Proc Natl Acad Sci USA 95:6578-6583, 1998
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This accounts for a biovolume of 1015 l
when a singele cell has a volume of 10-15 l
Mankind reaches only 5 · 1011 l
There are app. 5 · 1030 microorganisms in total
5 000 000 000 000 000 000 000 000 000 000
Parkes, R.J., B.A. Cragg and P. Wellsbury, 2000
Our main research interest:
The deep biosphere
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How to count cells in an environmental sample?
Methodological problems in microbial ecology:
- Only small morphological differences
- Cultivation efficiency often very low
How many bacterial species do we know?
Validly described species:
5 000 Prokaryotes (Bacteria and Archaea)
1 700 000 Eukaryotes
Estimation of bacterial species in 30 g soil
3 000 (Torsvik et al 1990, Appl Environ Microbiol 58:782-787)
based on the same data set:
500 000 (Dykhuizen 1998, Antonie van Leeuwenhoek 73:25-33)
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Phase-contrast microscopyCounting chamber
(Thoma, Petroff-Hausser, ...)
For liquid cultures and water samples only
Per
ry &
Sta
ley,
Mic
robi
olog
y –
Dyn
amic
s an
d D
iver
sity
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Filtration of water samples for epifluorescence microscopy
Staining of cells via DNA stains
Acridin Orange
Bacteria in a sediment particle
Pho
to: A
. Bat
zke
Pho
tos:
H. C
ypio
nka
Ironsulfide particle phase contrast
fluorescendcell
Combination ofphase contrast and
fluorescence
Other stains:
DAPI, SYBRgreen
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A novel protocol for SybrGreen-staining
Morono et al., 2009
Fluoresce In Situ Hybridisation
natural microbial
community
FixationTreatment with fixative, conditioning of cells,
filtration
WashingDetachment of probes that were not
bound to the target sequence
HybridisationAnnealing of probes under
stringent conditions
Fluorescent
dye
Specific
probes
16S rRNA
Counter stainingStaining of all cells by a general
fluorescent dye (e.g. DAPI)
VisualisationEpifluorescence microscopy
ProbeDAPI
Relation of non specific to specific signals
Fig
.: B
. Rin
k
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CAtalysed Reporter Deposition - FISH
natural microbial
community
FixationTreatment with fixative, conditioning of cells,
filtration
HybridisationAnnealing of probes under
stringent conditions
Horseradish-
peroxidase, HRP
Specific
probes
new
16S rRNA
WashingDetachment of probes that were not
bound to the target sequence
Fig
.: B
. Rin
k
Tyramide signal amplification (TSA)marked substrate (Tyramide) is enriched within the
cell by chemical reaction and binding to proteines
B
Protein
(Tyrosin)H2O
Peroxidase
H2O2
Activation
Enrichment
*
Peroxidase
H2O2
Fluorescent
dyeTyramide
inactiv
Anew
newWashing
Molecules that were not converted
Counter stainingStaining of all cells by a general
fluorescent dye (e.g. DAPI)
VisualisationEpifluorescence microscopy
ProbeDAPI
Relation of non specific to specific signals
Fig
.: B
. Rin
k
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Higher sensitivity by signal amplification
FISHCARD-FISH
dept
h (c
m)
Fig
.: M
. M
ussm
ann
Tidal flat sediment
Quantitative (real time) PCR
SybrGreen ITM-technique
⇒ almost no fluorescence ⇒ increasing fluorescence
Amplification
No binding onsingle stranded DNA
Intercalating of SybrGreen at double stranded DNA
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The apparatus
Rotor-Gene 2000/3000 Corbett Research, Australia
Raw data analysis
Rotor and detection unit
Quantitative PCR: Key Genes for metabolic pathways
MethanomicrobialesHydrogen + CO2
MethanosarcinalesMethylated aromates(humic acids, peat?)
MethanosarcinalesMethylamine, DMS
Wilms et al. (2007) FEMS Microbiol Ecol
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MPN-counts
Most Propable Number
Perry & Staley, Microbiology – Dynamics and Diversity
Colony forming units
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Slurry3
Slurry4
Control
ABC
DE
F
G
H
1 2 3 4 5 6 7 8 9 10 11 12
10 -1 10 -3 10 -5
10 -2 10 -4 10 -6
10 -6 10 -4 10 -2
10 -5 10 -3 10 -1
Slurry 1
Slurry 2
Control
Most Propable Number
MPN counts are strongly influenced by incubation conditions:
- Temperature
- Growth substrate
- Oxic or anoxic conditions
- Supply of additives and vitamines
Amino acids 1,9·107 cm3
Fatty acids 4,0·106 cm3
10°C 4,0·10 5 cm3
20°C 8,2·10 6 cm3
30°C 4,0·10 5 cm3
Oxic 1,0·107 cm3
Anoxic 4,0·105 cm3
MPN counts: tidal-flat sediments
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The borders of life:
Ecological range
What does an organism need for life
Water
Carbon source (org. C, CO2)
Engergy source (chemical Reaction)
Macro nutrients (N, P, S, Mg, Ca, Fe)
Trace elements (Mn, Co, Ni, W, Zn, Se, B, Mo, Cu)
A habitable milieu - ?
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Life conditions: Natural environment vs. laboratory
• Spatial heterogenieties
• In most cases no stable conditions: temporal variance (diurnal, annual)
• Substrate limitation (oligotrophic sites, selection k+r strategists)
• In most cases more than one substrate available
• Many types of organisms, competition, cooperation
Many reactions are only catalysed by prokaryontes:
- Nitrogen cycle (N2-fixation, etc.)
- Sulfur cycle (Sulfate reduction)
- Methanogenesis
- etc.
In principle, every biotope is inhabited by microorganisms
No ecosystem without microorganisms
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The role of microorganisms in the ecosystem
Exsamle:
Homo sapiens chemo organo heterotrophic
Chlorella sp. photo litho autotrophic
Thiobacillus sp. chemo litho autotrophic
Occurence in the ecosystem Concept of S.Winogradsky
Autochthonous or indigenic Typical, rel. stable population size
Allochthonous or zymogenic Untypical, strong population variancesfast growing
Chemo / Phototrophic Energy conservation
Litho / Organotrophic Electron donor
Auto / Heterotrophic Carbon source
Generalists – Specialists
Affiliation of organisms
Abb.: Fritsche (1999)
Electron and carbon source
Electron donor
Energy conservation
Redox condition
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Water content
Measure for the water content is the water activity aw
dest. water aw = 1 “normal” microorganisms aw = 0,9Seawater aw = 0,98 halophilic microorganisms aw = 0,75Salt lakes aw = 0,75 xerophilic fungy aw = 0,7
Problem: Osmolarity
Solution: Compatible Solutes (Osmolytica)in high concentration
Compatible solutes from hyperthermophilic microorganisms(Environ Microbiol 4:501ff)
e.g. Dimethylsulfoniopropionate (DMSP), Glycerine, Mannitol, Saccharose, Trehalose, Betain, Ectoin
Ectoin
Trehalose
CH3
CH3
H3C — N+ — CH3 — COOH
Betain
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Temperature
Life is dependant to liquid water.
Freezing point of seawater: -1,8°CAntarktic sea ice: -15°CSea ice has veins and cracks that still contain liquid water.
Middle oceanic ridges, geothermal sources
Due to over pressure: liquid water at 300 °C
The actual world record for hyperthermophiles is 121°C (doubtful)
(Kashefi & Lovley 2003 Science 301:934)
Prooven world record: 117°C (Stetter et al.)
Growth rates at different temperatures
... own investigations on a high-temperature oilfield
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Oil-bearinglayer
A subsurface sea of oil ?
5 µm
Scheme of the oil recovery system
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• Typical oil-related (hyper)thermophilic H2S-producing microorganisms
were detected in-situ and could be enriched and identified by DGGE
analysis.
• There were more H2S-producing microorganisms found in the pipelines
than in the production wells.
• The selective analysis of a biofilm showed, that most of the H2S-
producing microorganisms have settled at the pipline-walls.
• Variations in the community composition of the different sampling
campaigns showed a highly dynamic system and explains fluctuating
H2S-concentrations.
Conclusions
Fatty acids, that were found in bacterial lipids
saturated
iso-branching
ante iso-branching
unsaturated
alicyclic
Glycerol diether
Diglycerol tetraether
Rule of thumb: The higher the temperature,the more stable is the membrane.High content of saturated fatty acids.
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Pressure
... 1 bar pressure rise per 10 m; at 1000 m, pressure is a 100 times higher
Bacteria do not have a Schwimmblase.
Are bacteria pressure sensitive?
Experiment: Bring a balloon to a water depth of 1000 m
? ... or with water
?... filled with air °O (1 %)
O O (almost 100 %)
Barophilic microorganisms are adapted to high pressures e.g. higher amount of unsaturated fatty acids within their membrane, or modifed enzymes
However...
high pressures have an influence on:
- Boiling point and viscosity of water
- Membrane fluidity
- Stability of certain biomolecules