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

5 10 15 20 25

4104204304410523629729829929

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Depth

Temperature (C)

38.30 38.50 38.70 38.90 39.10

Salinity (psu)

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 Study site

CTD profile at Nestor 4.5

What is biofilm?

Abundance and diversity regulated by physico-chemical factors Bacteria prefer a sessile life formBiofilms most commonly form as a result of some stress

Therefore, biofilms are found in many extreme environments

Biofilm

(Davis , 2007)

Assemblage of microbial cells self-produced polymeric matrix

Within hours adhesion, colonization and growth of microbial populations

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

Objectives

• Biofilms on artificial substrata in deep-sea ?

• Bacteria in biofilms ?

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

Objectives

Characterization of :substrate relation within one deep-sea layerdepth relation for each substratedepth and substrate relation

Experimental Setup

Materials: titanium, aluminum, glass, limestone, shale

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

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

Methods

GKSS

Analyses method

Methods

Samples taken with a sterile

swap

DNA ExtractionPCR fluorescence (5’-FAM)

labeled primer

PCR product + Restriction enzymes

T-RFLP profiles

T-RFLP to compare microbial communities

Bacteria (Primer: 27F / 1492R)

Bacteria in biofilmsPCR

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

Negativecontrol

Results

Substrate relation within one depth - CfoI

Results

Substrate relation within one depth - RsaI

Results

Depth relation for each substrate - CfoI

Results

Depth relation for each substrate - RsaI

Results

Substrate vs depth - relation - CfoI

Results

Substrate vs depth - relation - RsaI

Results

Substrate vs depth - relation – Cfo I & RsaI

Results

Depth vs substrate relation - CfoI

Results

Depth vs substrate relation - RsaI

Results

Depth vs substrate relation – RsaI & CfoI

Results

Conclusions

PCR amplifications of 16S rDNA showed that DNA is verifiable: on each substrate

at all depth

Conclusions

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

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

Future perspectives

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’