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

1University of Milan, DISTAM, Via Celoria 2, 20133 Milan, Italy2Tecnomare s.p.a., San Marco 3584, 30124 Venice, Italy

3University of Milan-Bicocca, DGSG, P.zza della Scienza 4, 20126 Milan,

Italy

DHABs: extreme environments

3200-3700 m deep

High salinity (>30%)

Steep chemocline (O2, H2S, 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 UraniaL’AtalanteDiscovery

Do microbes inhabit DHABs?

BannockUraniaL’AtalanteDiscovery

BIODEEP projectBIOtechnologies from the

DEEP

BIODEEP cruisesR/V URANIA

•August 2001•June 2002

DAPI cell counts

Disco

very

Uran

ia

L’At

alan

te

Bann

ockcells/ml of sample

1500m

2500m

3300m

2,8 104

4,3 104

3,9 104 2,3 104

6,1 105 3,4 106 1,2 106 1 106

1,9 104 1,5 105 7,8 104 4,7 104

Water column

Water/brines interface

brines

W 1500m

B 3500m

W 2500m

W 3300m

WB

ARDRAeubacteria

RISAeubacteria

DGGEarchaea

inte

rface

A B U D

brin

ein

terfa

ce

brin

ein

terfa

ce

brin

ein

terfa

ce

inte

rface

brin

ein

terfa

ce

brin

ein

terfa

ce

inte

rface

U U A A B D B A U D B A U

brines interfaces

basin

Typing of Eubacteria and Archaea in DHABs

Peculiar communities inhabit DHABs

Bannock

Urania

L’AtalanteD

isco

very

Upper seawater column

PC 1 (29,5% of the variability)

PC

2 (

10%

of

the v

ari

ab

ility

)

B

I

I

I

I

BB

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

3521.5m 3523.5m

Con

du

ctiv

ity (

salin

ity)

CTD deployment

CTD recovery

dep

th

WATER

BRINES

UR

AN

IA I

NTER

FA

CE10L Niskin bottle n°3

10L Niskin bottle n°4

Closure of Niskin bottles vs. depth

The chemocline1

0 L

Nis

kin

bott

le

1st litre

2nd litre

3rd litre

4th litre

10th litre

….

Niskin 3

Seawater

Brine

Niskin 4

1 2 3 4 5 6 7 8 9 10

con

du

ctiv

ity 1 2

34

5 6 7 8 9 10

1.02

1.03

1.04

1.05

1.06

1.07

1.08

1.09

1.1

1.11

1.12

30.00 50.00 70.00 90.00 110.00 130.00 150.00

0

20

40

60

80

100

120

140

30.00 50.00 70.00 90.00 110.00 130.00 150.00

0

20

40

60

80

100

120

140

30.00 50.00 70.00 90.00 110.00 130.00 150.000

20

40

60

80

100

120

140

30.00 50.00 70.00 90.00 110.00 130.00 150.00

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 3 Niskin 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 bandsDisappearing bands

0

20

40

60

80

100

120

140

3,7

5,7

12,8

15,4

16,5

17,7

18,4

18,6

21,1

salinity %

DN

A (

ng

/ml)

1,E+05

1,E+06

1,E+07

cell

s/m

l

DNA (ng/ml)

cells/ml

DAPI cells countsandDNA extraction yields

URANIA INTERFACEB1 B2

Along the salinity gradient are there quantitative differences among microbial

populations?

TaqMan Real time PCR targeting 16S rDNA:universal for prokariotesuniversal for archaea

archaea and eubacteria cells

1.E+03

1.E+04

1.E+05

1.E+06

1.E+07

40 60 80 100 120

conductivity

cell

s archaea cells

eubacteria cells

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

http://www.geo.unimib.it/BioDeep/Project.html

BIODEEP projectBIOtechnologies from the DEEP

Thanks to:the scientific and technical staff, and the crew of the Urania

oceanographic ship.