1. Bacterial communities in the seawater-brine chemocline in deep anoxic hypersaline basins (DHABs) in the eastern Mediterranean Sea Borin S., Daffonchio D., Brusa T., Gasparoni F.,Calore D., Corselli C., and BIODEEP Scientific Team 1 University of Milan, DISTAM, Via Celoria 2, 20133 Milan, Italy 2 Tecnomare s.p.a., San Marco 3584, 30124 Venice, Italy 3 University of Milan-Bicocca, DGSG, P.zza della Scienza 4, 20126 Milan, Italy

2. DHABs: extreme environments 3200-3700 m deep High salinity (>30%) Steep chemocline (O 2, H 2 S, salinity) at the sea water/brine interface Absence of light High pressure Anoxic High density: no mix with overlaying sea water Very old origin (Messinian evaporites dissolution) Bannock Urania L’Atalante Discovery  Do microbes inhabit DHABs?

3. Bannock Urania L’Atalante Discovery BIODEEP project BIOtechnologies from the DEEP BIODEEP cruises R/V URANIA August 2001 June 2002

4. DAPI cell counts Discovery Urania L’Atalante Bannock cells/ml of sample 1500m 2500m 3300m 2,8 10 4 4,3 10 4 3,9 10 4 2,3 10 4 6,1 10 5 3,4 10 6 1,2 10 6 1 10 6 1,9 10 4 1,5 10 5 7,8 10 4 4,7 10 4 Water column Water/brines interface brines W 1500m B 3500m W 2500m W 3300m WB

5. ARDRA eubacteria RISA eubacteria DGGE archaea interface ABUD brine interface brine interface brine interface brine interface brine interface UUAABDBAUDBAU brinesinterfaces basin Typing of Eubacteria and Archaea in DHABs

6. Peculiar communities inhabit DHABs Bannock Urania L’Atalante Discovery Upper seawater column PC 1 (29,5% of the variability) PC 2 (10% of the variability) B I I I I B B Principal Component Analysis of eubacterial community RISA profiles

7. Sea water/brines interfaces act as “particle traps” and are an enrichment phase for bacteria Study of the chemocline Community fingerprinting DAPI counts Total DNA quantification Real time PCR Prokariotes and Archaea

8. Deep Sea DHABs Sea floor MODUS Accurate chemocline sampling R/V Urania SciPack Remote operated MODUS/SCIPACK system developed to collect water and brine samples with an accurate depth and position control in the DHABs environment. Biodeep cruise, august 2001 S/W interface

9. Conductivity vs. depth: Urania sea water/brine interface Seawater Brines 3521.5m3523.5m Conductivity (  salinity) CTD deployment CTD recovery

10. depth WATER BRINES URANIA INTERFACE 10L Niskin bottle n°3 10L Niskin bottle n°4 Closure of Niskin bottles vs. depth

11. The chemocline 10 L Niskin bottle 1 st litre 2 nd litre 3 rd litre 4 th litre 10 th litre …. Niskin 3 Sea water Brine Niskin 4 12 3 4 5 6 7 8 9 10 conductivity 1 2 3 4 5 6 7 8 9 10 Density (g/l) HS- (  moles/l) Urania basin Bannock basin DNA extraction and microbial community molecular characterisation

12. Water/brine interface: eubacterial diversity along the salinity gradient Niskin 3Niskin 4 ARDRA eubacteria ARISA eubacteria Salinity: 3.7% Salinity: 5.1% Salinity: 5.7% Salinity: 9.1% Salinity: 16.1% Using sensitive techniques such as ARISA some population shifts are observed New bands Disappearing bands

13. DAPI cells counts and DNA extraction yields URANIA INTERFACE B1 B2 Along the salinity gradient are there quantitative differences among microbial populations? TaqMan Real time PCR targeting 16S rDNA:  universal for prokariotes  universal for archaea

14. Bacteria in the DHABs chemoclines Sea water/brine interface seems an enrichment phase especially for Eubacteria Diversity shifts can be observed along the chemocline of Urania basin Microbial community in the chemocline at the sea water/brine interface is different from the brine and the upper sea water Chemoclines at the sea water/brine interfaces act as particle trap Microbial communities in the different DHABs are very diverse

15. funded by the European Union Marine Science and Technology programme Fifth Framework Programme http://www.geo.unimib.it/BioDeep/Project.html BIODEEP project BIOtechnologies from the DEEP Thanks to: the scientific and technical staff, and the crew of the Urania oceanographic ship.