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Microbial ecology

VL 1

Microbial abundance

and ecological range

Bert Engelen engelen@icbm.de

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

9

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