Work package 3Analysis of the species diversity,

community structure and phylogeny of microorganisms.

Laboratory of Microbial Ecology

CEES/RUG

Objectives• Evaluate microbial communities in 4 deep sea

hypersaline anoxic basins (DHABs)

• Evaluate similarities between DHABs and between different positions in one DHAB

• Link previously published geochemical data with these results

Brine interface waterFilter

DNA-isolation

Bacterial 16S rDNA

PCR

Archaeal 16S rDNA

PCR

Cloning PCR-product

Sequencing 700 bases

Sequencing 700 bases

Cloning PCR-product

Bacteria

Brine Interface Seawater

Atalante Bannock Discovery Urania Atalante Bannock Discovery Urania Bannock Discovery

Sequences 90 90 90 90 90 90 90 90 90 90

OTUa 40 41 48 16 28 27 36 25 39 57

Shannon 3.26 3.20 3.51 2.17 2.90 2.72 3.27 2.78 3.42 3.88

Archaea

Brine Interface Seawater

Atalante Bannock Discovery Urania Atalante Bannock Discovery Urania Bannock Discovery

Sequences 90 90 90 90 90 90 90 90 90 90

OTU 11 10 10 5 15 14 15 16 17 12

Shannon 1.60 1.49 1.55 0.61 2.01 1.83 1.77 1.44 2.30 1.87

PCoA of Morisita’s Index for Bacterial OTU-level

17.9

% o

f V

aria

nce

1 9 .9% o f Var ia n ce

C o ord in ate 1

Coo

rdin

ate

2

U IB I

D I

A IB B

A B

D B

D 3 30 0

B 3 00 0

U B

0 .30 .20 .10-0.1-0.2-0.3 0 .60 .50 .4

-0.3

0 .1

0 .2

0 .6

-0.2

0-0.1

0 .4

0 .5

0 .7

0 .3

PCoA of Morisita’s Index for Archaeal OTU-level

U B

18.2

% o

f V

aria

nce

5 1 .5% o f Var ia n ce

C o ord in ate 1

Coo

rdin

ate

2

-0.4 0.40 .30 .20 .10-0.1-0.2-0.3

-0.4

0 .4

0 .8

0 .2

0 .6

-0.2

0

U I

B I

D I

A I B BA B

D B

D 3 30 0

B 3 00 0

Cluster Analysis (UPMGA) of Morisita’s Index for OTU-level

Bacterial Brine+Interface

Archaeal Brine

Archaeal Interface

BB AB DBUB

0.2

0.6

0.4

0.8

1.0

PCoA of Morisita’s Index for Bacterial Phylogenetic Group-level

23.3

% o

f V

aria

nce

4 5 .3% o f Var ia n ce

C o ord in ate 1

Coo

rdin

ate

2

U I

B I

D I

A I

B B

A BD B

D 3 30 0

B 3 00 0

U B

0 .30 .20 .10-0.1-0.2-0.3 0 .50 .4

-0.3

0 .1

0 .2

-0.2

0

-0.1

0 .4

0 .3

-0.4

-0.4

-0.5

0

7.6%

of

Var

ianc

e

8 8 .0% o f Var ia n ce

C o ord in ate 1

Coo

rdin

ate

2

U I

B I

D I

A IB B

A B

D B

D 3 30 0B 3 00 0 U B

0 .30 .20 .10-0.1-0.2-0.3-0.4

0 .2

0 .6

-0.2

0 .4

0 .8

PCoA of Morisita’s Index for Archaeal Phylogenetic Group-level

Principal Component Analysis

• The two seawater samples always group together

• Discovery brine and Discovery Interface always group together

• L’Atalante, Urania and Bannock brines group together except for Bannock Bacterial

• L’Atalante, Urania and Bannock interface group together except for L’Atalante Archaea

• In most cases, Discovery samples group together with other brine samples

Cluster analysis• Discovery is most different from the

other DHABs for the bacterial and archaeal brine and the bacterial interface samples.

• Next to Discovery, Urania is most different from the other DHABs

• Two clusters were observed for the archaeal interface samples

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

delt

a-pr

oteo

Bac

tero

idet

es

epsi

lon

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new

/unk

now

n5 ga

mm

a-pr

oteo

KB

1

AB

BB

DB

UB

AI

BI

DI

UI

B3000

D3300

Percentage (%)

Relative Distribution of Phylogenetic Bacterial Groups

0

10

20

30

40

50

60

70

80

90

100

Marine Group I MSBL1 Halobacteriales

AB

BB

DB

UB

AI

BI

DI

UI

B3000

D3300

Percentage (%)

Relative Distribution of Phylogenetic Archaeal Groups

Metabolic Routes

- High number of δ-proteobacteria Sulfate reduction Important

H2, organic matter or anaerobic CH4 oxid.

No archaea related to anaerobic CH4 oxid.

δ-13C CO2 values are not extremely low

H2 and organic matter most important for SO4-reduction

Metabolic Routes

• ε- and γ-proteobacteria were mainly related to sulfide oxidizing bacteria.

• These sequences were most numerous in Urania

• Sulfide oxidation plays an important role in the interface samples

• High number of Bacteroidetes shows that heterotrophic bacteria play important role

Archaea

• Archaea related to Marine group I and a new cluster MSBL1

• At present unknown what the function is of these archaea

• Discovery high number of Halorhabdus utahensis

• This bacterium adapted to high concentrations of Mg2+

• Interface samples different from brine samples. The sharp increase in salinity, sulfide and decrease in O2 are very important factors for the establishment of a microbial community.

• Discovery different. MgCl2 is thus important factor in establishment of microbial community

• Next to Discovery, Urania differs. Sulfide concentration is an important factor as well.

Conclusions

Conclusions• Microbial routes present in the sulfur cycle

have an important function in the DHABs

• Most of the archaeal sequences form a new branch in the archaea tree. This cluster is named MSBL1

• The function of almost all archaea as well for a great number of bacteria is at present unknown

2 A2 B

1 A1 B

3 A

3 B

4 B4 A

33.3

% o

f V

aria

nce

4 5 .5% o f Var ia n ce

Bannock interface samples cruise II

Future Plans• A complete phylogenetic analysis with

GenBank database of the Bannock interface samples

• Study temporal variation by comparing sequences from brines of cruise I, II (and III?)

• Study diversity of several functional genes or specific groups with specific 16S rDNA primers