Laboratory of Microbial Ecology CEES/RUG

1. Work package 3Analysis of the species diversity, community structure and phylogeny of microorganisms. Laboratory of Microbial Ecology CEES/RUG

2. 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

3. Brine interface water Filter DNA-isolation Bacterial 16S rDNA PCR Archaeal 16S rDNA PCR Cloning PCR-product Cloning PCR-product Sequencing 700 bases Sequencing 700 bases

5. PCoA of Morisita’s Index for Bacterial OTU-level

6. PCoA of Morisita’s Index for Archaeal OTU-level

7. UB AB DB BB 1.0 0.8 0.6 0.4 0.2 Cluster Analysis (UPMGA) of Morisita’s Index for OTU-level Bacterial Brine+Interface Archaeal Brine Archaeal Interface

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

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

10. 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

11. 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

12. Relative Distribution of Phylogenetic Bacterial Groups Percentage (%)

13. Relative Distribution of Phylogenetic Archaeal Groups Percentage (%)

14. 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

15. 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

16. 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+

17. Conclusions • 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.

18. 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

19. Bannock interface samples cruise II

20. 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