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, Milan, Italy 2 Tecnomare s.p.a., San Marco 3584, Venice, Italy 3 University of Milan-Bicocca, DGSG, P.zza della Scienza 4, Milan, Italy

DHABs: extreme environments 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?

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

DAPI cell counts Discovery Urania L’Atalante Bannock cells/ml of sample 1500m 2500m 3300m 2, , , , , , , , , , , Water column Water/brines interface brines W 1500m B 3500m W 2500m W 3300m WB

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

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

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

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

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

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

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 conductivity Density (g/l) HS- (  moles/l) Urania basin Bannock basin DNA extraction and microbial community molecular characterisation

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

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

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

funded by the European Union Marine Science and Technology programme Fifth Framework Programme BIODEEP project BIOtechnologies from the DEEP Thanks to: the scientific and technical staff, and the crew of the Urania oceanographic ship.