Tingting Yang, Lisa Nigro, Luke Mckay, Tony Gutierrez, Andreas Teske UNC Chapel Hill Oct-27-2010

1. Microbial community dynamics of the oil leak: time series study, microbial water column stratification, hydrocarbon substrate response Tingting Yang, Lisa Nigro, Luke Mckay, Tony Gutierrez, Andreas Teske UNC Chapel Hill Oct-27-2010

2. Molecular microbiological experiment • Cultivation experiment

3. Molecular microbiology: Bacterial 16S rRNA gene clone libraries • Water samples are from 3 cruises: • RV Pelican cruise (May 4th-16th) • Oily surface water sample • RV Walton Smith cruise (May 26th-June 8th) • Plume water and water from above and below plume layers • RV Pelican cruise (September 9th-15th) • Same site and depth as Walton Smith cruise, after plume has gone. • All water samples (500 ml) were filtered through 0.22 µm Anodisc filters or ISOPORE membrane filters.

4. Pelican cruise (May 4th-16th)--Surface oily water samples

5. Cycloclasticus is a special polycyclic aromatic hydrocarbon (PAH)-degrading bacteria “Gulf of Mexico Cycloclasticus strains had broad substrate ranges which included naphthalene, substituted naphthalenes, phenanthrene, biphenyl, anthracene, acenaphthene, and fluorene.” (Geiselbrecht et. al, 1998)

6. “We suggest that the observed relatives of Cycloclasticus and/or Colwellia are blooming as a result of their capacity to consume propane, ethane, and potentially butane, although not at the exclusion of other bacteria or metabolisms. While Cycloclasticus is known for its ability to degrade aromatic compounds, sequences observed here are 90% similar to putative ethane and propane oxidizers identified by stable isotope probing, indicating the capability in this evolutionary lineage.”

7. Walton Smith cruise (May 26th – June 8th) --Deep plume water samples Plume water 1100-1250 m

8. 800 m 1170 m 1210 m 1320 m

9. Phylogenetic tree of plume samples (1170 m and 1210 m) The close relatives are aerobic alkane oxidizers of the genera Oleispira and Thalassolituus.

10. Near-bottom water sample below plume (1320 m) Sea water sample above plume (800 m)

11. Pelican cruise (Sep 9th-15th)-- Water sample from former plume site • We went back to old sites where plume was found in Walton Smith cruise. • No plume any more.

12. It looks like the microbial diversity at the plume site has recovered. To be checked with statistical comparison of above-plume sample (May 31) and post-plume sample (Sept 9-16)

13. Conclusion of molecular microbial experiment Effect of oil spill on microbial community • Surface (0 m) - Dominated by Cycloclasticus group (Gammaproteobacteria); • Medium sea water (800 m) - High diversity Alphaproteobacteria are the main group • Plume water (1170 m & 1210 m) - Dominated by Oceanospirillales group (Gammaproteobacteria) • Deep water (1310 m) - High diversity, high proportion of Alphaproteobacteria and Deltaproteobacteria • Time series study shows the microbial diversity at the plume site has increased (methylotrophs, aromatic oxidizers and typical marine bacteria).

14. Cultivation experiment • Two methods • Series dilution • Direct isolation

15. Plume Water 0.5ml 0.1 ml 50 µl 10 µl 1 µl 0.01 µl 100 µl Hexadecane + 3 ml ONR medium Room temperature Shake in dark Series dilution

16. 3rd day

17. Check whether hexadecane is the only C source of these cultures. Get pure cultures PCR & Sequencing

18. Direct isolation Plume Water 100 µl 50 µl 25 µl 10 µl 5 µl + ONR plates No hexadecane plates Hexadecane

20. Alcanivorax spp alkane degrader （http://confrontaal.org/wordpress/?p=7914）

21. First RV Pelican cruise w. Arne Diercks: Luke Mckay • RV Walton Smith cruise w. Mandy Joye: Lisa Nigro and Tingting Yang • Second RV Pelican cruise w. Ken Sleeper: Tingting Yang, Howard Mendlovitz and Dan Hoer • RV Cape Hatteras cruise w. Kevin Yaeger: Kelly Speare (just came back with oily sediment samples!) Thanks Andreas Teske, Mandy Joye, Tony Gutierrez, Kai Ziervogel, Lisa Nigro, Luke Mckay, Barbara MacGregor, Kelly Speare!