1. Exploring the Effects of Hypoxia on Sulfate Reducing Anaerobes Annaliese Jones Project Advisor: Bethany Jenkins

2. What is Environmental Microbiology? Most recent estimate: 5 million trillion trillion (5x10 30 ) The study of microbes in the environment that affect earth and it’s atmosphere. physiological activity that affects the environment (and us).

3. Microbial Activities Decomposition Generate O 2 in atmosphere Symbiotic relationships with plants and animals – Bacterial cells outnumber human cells 10:1. Global cycling of nutrients

4. Decreasing Energy Bacteria can “breathe” without Oxygen O 2 Fe +3 NO 2 - NO 3 - SO 4 -2 S 2H + 2H 2 O Fe +2 NH 4 + NO 2 - H 2 S H 2

5. When can environments run out of oxygen?

9. Hypoxia in Our Own Backyard >2 mg O 2 / L H 2 O

10. Increasing Depth Overlying Water Sediment Surface Sulfate Reducers Hypoxia creates a niche for bacteria that “breathe” other substances Previous work in Jenkins lab shows N fixation by S and SO 4 -2 reducers. Examine link between Hypoxia,N fixation, SO 4 -2 reduction. O 2 Fe +3 NO 2 - NO 3 - SO 4 -2 S 2H + 2H 2 O Fe +2 NH 4 + NO 2 - H 2 S H 2

11. 4 4 31 Sample Sites: Previously Examined for N fixation = Midbay Site Greenwich Bay Sites

12. 2.5cm DNA  Who’s there RNA  Who’s expressing 2.5cm DNA RNA Whole Round Core Sampling Microbes in Marine Sediment

13. Gel Electrophore sis Cloning AGTCAAACTGCATGG Sequence data Where do I belong? Extraction Environmental Genetic Material DNA RNA cDNA PCR Reaction Cycle 1 2 Amplifying Genes of Interest from the Environment Copies of Gene of Interest: dsrB, nifH

14. Which Genes are being Followed in the Bay? nifH dsrB Dinitrogenase reductase Part of enzyme responsible for bacterial N fixation (nitrogenase) N 2  NH 4 + Dissimilatory sulfite reductase Enzyme involved in SO 4 -2 reduction pathway. SO 4 -2  H 2 S AGTCAAACTGCATGG Sequence data Where do I belong?

15. Phylogenetic Tree = “Family Tree” Taxon A Taxon C Taxon B Taxon D Taxon E Taxon= “Family member” genes, populations, species. Building a Database of Sequences Collections of sequence data available online. Download sequences for gene(s) of interest. Compile your own database with those particular genes. Use your database to figure out where your samples belong. This length matters

16. “Family Tree” of dsrB Black text= cultivated species Colored text= Narragansett Bay sequences = Site 1 = Site 3 = Site 4 = Midbay

17. (gutless marine worm) Delta 1 endosymbiont. Live in symbiosis with sulfate oxidizing bacteria. Previously only studied in Mediterranean. Lives in low O 2. Sediments. Olavius algarvensis, Olavius ilvae

18. Isolated from a water-oil separation system in the North Sea. Capable of fixing nitrogen. Relative of Desulfobacter latus, which previously appeared in nifH tree. Resistant to high concentrations of heavy metals (Fe, Cu, Zn). Subject of ongoing research for use in bioremediation. Used in acid mine drainage decontamination. Isolated from water-oil separation system in North Sea. Frequently found in wastewater treatment sites. Desulfobacter vibrioformis Desulfobulbus rhabdoformis

19. Site 1: dsrB Present Regardless of O 2 1 dsrB Presence May 2010 July 2010 June 2011 nifH Expression SO 4 -2 reducer presence does not vary significantly given similar DO conditions. No observable correlation between presence of SO 4 -2 reducers and nifH expression ( N 2 fixation). Very diverse. Contains Olavius endosymbiont relatives.

20. Site 3: dsrB expression correlates with hypoxia 3 May 2010 July 2010 dsrB Presence dsrB Expression nifH Expression Possible correlation between hypoxia and active SO 4 -2 reduction. Spatial overlap. Sequences align with sulfate reducers capable of fixing N 2.

21. 4 Site 4: dsrB presence not widespread dsrB Expression nifH Expression May 2010 July 2010 June 2011 dsrB Presence Sulfate reduction not widespread in sediment. No overlap between dsrB expression and nifH expression. Consistent with previous data indicating increased # of transcripts for N fixing sulfur reducers. Given similar DO conditions, presence of SO 4 -2 reducers varies significantly.

22. Next Steps Gather Site 1 dsrB expression data for 2010. -Determine if there is a correlation between hypoxia and dsrB expression at this site. Gather Site 3 dsrB presence and expression data for 2011. -Examine variance in presence. Quantitative PCR to determine activity levels for each site. Broad Conclusions Site 3 demonstrates a high degree of correlation between active SO 4 -2 reduction and active N 2 fixation. Site 4 demonstrates a correlation between dsrB expression and hypoxia. Shows similar pattern to previous nifH data. Possible additional factors affecting dsrB expression and presence/absence of sulfate reducing bacteria: - Difference in sediment grain consistencies between sites -Proximity to wastewater treatment center (source of organic matter and nutrients).

23. Acknowledgements Dr. Bethany Jenkins Shelley Brown Rodrigue Spinette Andraya Ehrlich Dreux Chappell Leann Pritchard Samua Jean-Charles Christopher Deacutis-RI DEM URI Honors College Office of the Provost National Science Foundation References Heidelburg, J.F et al. The genome sequence of the anaerobic, sulfate- reducing bacterium Desulfovibrio vulgaris Hildenborough. Nature Biotenchnology.22. 554-559. (2004). Dar, S. A., Yao, L., Kuenen, G.J., and Muyzer,G. Analysis of Diversity and Activity of Sulfate-Reducing Bacterial Communities in Sulfidogenic Bioreactors Using 16S rRNA and dsrB Genes as Molecular Markers. Environmental Biotechnology. 73. 594-604. (2007). Sievert, S.M., Kiene, R.P., Schulz-Vogt, H.N. The Sulfur Cycle. Oceanography. 20. 117-123 (2007)