1. 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

2. 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/cm 3  slow accumulation rate of 7– 8cm over 10000 years Study site CTD profile at Nestor 4.5

3. What is biofilm? 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 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)

4. 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 layer depth relation for each substrate depth and substrate relation

5. Experimental Setup Materials: titanium, aluminum, glass, limestone, shale Depths: 1500m, 2500m, 3500m, 4500m Exp. duration: May - October 2007 (R/V Aegaeo) Methods GKSS

6. Analyses method Methods Samples taken with a sterile swap DNA Extraction PCR fluorescence (5’-FAM) labeled primer PCR product + Restriction enzymes T-RFLP profiles T-RFLP to compare microbial communities

7. Bacteria (Primer: 27F / 1492R) Bacteria in biofilms PCR H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 Negativ e control Results

8. Substrate relation within one depth - CfoI Results

9. Substrate relation within one depth - RsaI Results

10. Depth relation for each substrate - CfoI Results

11. Depth relation for each substrate - RsaI Results

12. Substrate vs depth - relation - CfoI Results

13. Substrate vs depth - relation - RsaI Results

14. Substrate vs depth - relation – Cfo I & RsaI Results

15. Depth vs substrate relation - CfoI Results

16. Depth vs substrate relation - RsaI Results

17. Depth vs substrate relation – RsaI & CfoI Results

18. 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

19. 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

20. 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’