Use of T-RFLPs for analyzing substrate attached bacteria in biofilms
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Use of T-RFLPs for analyzing substrate attached bacteria in biofilms in the deep-waters of the Hellenic Trench (Ionian Sea). Christian-Albrechts-University of Kiel , FTZ, Hafentörn 1, Büsum, Germany Hellenic Centre for Marine Research , 46km Athens-Sounio, Anabyssos, Greece.

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Christian albrechts university of kiel ftz hafent rn 1 b sum germany

Use of T-RFLPs for analyzing substrate attached bacteria in biofilms in the deep-waters of the Hellenic Trench (Ionian Sea)

Christian-Albrechts-University of Kiel, FTZ, Hafentörn 1, Büsum, Germany

Hellenic Centre for Marine Research , 46km Athens-Sounio, Anabyssos, Greece

Nikoleta Bellou, Evangelos Papathanassiou and Franciscus Colijn


Study site

Study Site

Salinity

Temperature

Study site

Deepest part of the Mediterranean

Lies far from the effluents of major rivers

extremely clear water (light transmission length

55 + 10 m at 460 nm wavelength)

Weak water current consistently (below 10 cm/s)

Mean density of sea floor sediments 1.5 gr/cm3

slow accumulation rate of 7– 8cm over 10000 years

CTD profile at Nestor 4.5


What is biofilm

Biofilm

What is biofilm?

First introduced by Zobell & Allen (1935) and Zobell & Anderson (1937)

Abundance and diversity regulated by physico-chemical factors

Bacteria prefer a sessile life form

Biofilms most commonly form as a result of some stress

Therefore, biofilms are found in many extreme environments

Assemblage of microbialcells

self-produced polymeric matrix

Within hours adhesion, colonization and growth of microbial populations

(Davis , 2007)


Objectives

Objectives

Objectives

Biofilms on artificial substrata in deep-sea ?

Bacteria in biofilms ?

Comparison microbial communities in biofilms grown on different substrates & grown in different depths

Characterization of :

substrate relation within one deep-sea layer

depth relation for each substrate

depth and substrate relation


Experimental setup

Methods

Experimental Setup

Materials: titanium, aluminum, glass, limestone, shale

Depths: 1500m, 2500m, 3500m, 4500m

Exp. duration: May - October 2007 (R/V Aegaeo)

GKSS


Analyses method

Analyses method

Methods

T-RFLP to compare microbial communities

DNA Extraction

PCR product + Restriction enzymes

PCR fluorescence (5’-FAM) labeled primer

Samples taken with a sterile swap

T-RFLP profiles


Bacteria in biofilms pcr

Results

Bacteria in biofilmsPCR

Bacteria

(Primer: 27F / 1492R)

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20

H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20

Negative

control


Substrate relation within one depth cfoi

Substrate relation within one depth - CfoI

Results


Substrate relation within one depth rsai

Substrate relation within one depth - RsaI

Results


Depth relation for each substrate cfoi

Results

Depth relation for each substrate - CfoI


Depth relation for each substrate rsai

Depth relation for each substrate - RsaI

Results


Substrate vs depth relation cfoi

Substrate vs depth - relation - CfoI

Results


Substrate vs depth relation rsai

Substrate vs depth - relation - RsaI

Results


Substrate vs depth relation cfo i rsai

Substrate vs depth - relation – Cfo I & RsaI

Results


Depth vs substrate relation cfoi

Depth vs substrate relation - CfoI

Results


Depth vs substrate relation rsai

Depth vs substrate relation - RsaI

Results


Depth vs substrate relation rsai cfoi

Depth vs substrate relation – RsaI & CfoI

Results


Conclusions

Conclusions

Conclusions

PCR amplifications of 16S rDNA showed that DNAisverifiable:

on each substrate

at all depth

Bacterial biofilm communities

do not differ highly between substrates within one depth

BUT differ along a depth gradient in one substrate type

Results indicate highly stratified biofilm communities


Future perspectives

Future perspectives

Future perspectives

Characterization of biofilms community structure collected from

other deep sea Mediterranean sites

Compare substrate attached bacteria (biofilm)

with particulate attached (sediment traps)

with free living (water column)

Biochemical characterization of biofilm


Christian albrechts university of kiel ftz hafent rn 1 b sum germany

THANK YOU !Thanks to:Vassilis Lykoussis (HCMR / Greece)Aleka Gogou (HCMR/ Greece)Voigt Wolfgang (CAU / Germany) Ruediger Kiehn (GKSS / Germany)Torsten Staller (CAU / Germany)Elena Sarropoulou (HCMR / Greece)Katerina Skaraki(HCMR / Greece)

6thFramework Program of the European Commission to support the ‘European Research Area’


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