christian-albrechts-university of kiel , ftz, hafentörn 1, büsum, germany
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
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
102911291229133114331533163417341834193420342134223423342434253426342734283429343034313432343337343735373638373938393988408841894289
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’