1. Microbiology of Sulfate- Reducing Passive Treatment Systems Amy Pruden 2, Sage R. Hiibel 1, Luciana P. Pereyra 2, Laura Y. Inmann 1, Nella Kashani 1, Kenneth F. Reardon 1, and David Reisman 3 1 Chemical and Biological Engineering Department, Colorado State University 2 Civil and Environmental Engineering Department, Colorado State University 3 U.S. Environmental Protection Agency

2. What is Mining-Influenced Water (MIW)? When water discharges to surface: 4 Fe +2 + O 2 + 10 H 2 O  4 Fe(OH) 3(S) + 8 H + Yellow Boy 4Fe 2+ (aq) + 8SO 4 2- (aq) + 8H + (aq)

3. Using Sulfate-Reducing Permeable Reactive Zones (SR-PRZs) to treat MIW

4. Conceptual SR-PRZ Microbial Community Methanogenesis CH 4 CO 2 Sulfate reduction HCO 3 - H 2 S Enzymatic hydrolysis Fermentation H2H2 CO 2

5. Objective Conduct a biomolecular survey of microbial communities in passive treatment systems in the U.S.

6. Field Sites

7. Luttrell SR-PRZ Sampled June 13, 2005 Inlet Outlet Inlet Outlet Impervious Liner Plane View Side View Sampling Location Top = 1-11 cm below surface Bottom = 15-25 cm below surface

8. Peerless Jenny King SR-PRZ Sampled June 14 & August 30, 2005 Sampling Locations Top = 1 – 11 cm below surface Bottom = Just above rock substrate

9. Field Site Design Comparison

10. Molecular Techniques Utilized Microbial Community Profiling Qualitative Techniques: Cloning Total Bacteria Sulfate Reducing Bacteria (SRB) SRB quantification Quantitative Techniques: Quantitative PCR Desulfovibrio spp. Desulfobacterium spp. Total Bacteria

11. DNA Extraction Community DNA 16S or apsA Gene PCR Amplification Community 16S rDNA or apsA genes Ligate to vector Transform into E. coli Plate and grow cells Screen Clones, Sequence DNA, ID Bacteria Sample Molecular Techniques Utilized Community Profiling

12. Real-time Q-PCR: Quantify amount of DNA initially present…… Fluorescence Number of PCR Cycles Threshold Cycle (C T ) increases as initial [DNA] decreases Molecular Techniques Utilized SRB Quantification

13. Results Cloning: Diversity Comparison (June)

14. Fermentation Cellulose Degradation Mn Reduction Sulfate Reduction Hydrocarbon Degradation Iron Reduction Nitrogen Fixation Methane Degradation Unknown Nitrate Reduction Cloning: 16S Gene Sequencing

15. Desulfohalobium retbaense Desulfovibrio alkalitolerans Desulfovibrio burkinensis Desulfovibrio termitidis Desulfovibrio spp. Thiobacillus denitrificans Uncultured SRB Desulfovibrio aerotolerans Desulfovibrio africanus Cloning: apsA Gene Sequencing (Targets SRB)

16. LUTR PJK LUTR Bottom LUTR Top LUTR 2 Top PJK 1i Bottom PJK 3 Bottom PJK 3 Top Results QPCR: SRB Quantification

17. Conclusions Luttrell SR-PRZ –Higher variety of putative function in top region Mn and N reducing bacteria indicative of less reducing environments than typical of fermentation environments –Verified by presence of D. aerotolerans –SRB communities predominately D. burkinensis –Oxygen-tolerant SRB noted in top region 10% D. aerotolerans

18. Conclusions Peerless Jenny King SR-PRZ –Distinctly different community than Luttrell –Distinctly different communities in each location and each bioreactor –More putative functions than LUTR in all sampling locations –apsA analysis showed predominately T. denitrificans and D. burkinensis Oxygen exposure?

19. Future Work Compare 2005 and 2006 microbial communities in PJK Continue to link microbial community with performance –Improve understanding of microbial role in remediation Target 16S rRNA –Lifetime of rRNA <<< rDNA –Indication of microorganisms active in community Target additional functional genes –Determine which processes are occurring

20. Acknowledgements David Reisman – EPA Robb Amick – EQ Management, Inc. Jim Gusek – Golder Associates Linda Figueroa – Colorado School of Mines EPA Hazardous Substance Research Center (EPA STAR Grant R 8395101-0)

21. QUESTIONS? THANK YOU

22. Polymerase Chain Reaction (PCR) Amplifies sequences of DNA using target- specific primers and thermal cycling

23. Cloning Allows mixed microbial communities to be separated by growth on an antibiotic- impregnated plate

24. Cloning - Vector

25. LUTR PJK LUTR Bottom LUTR Top LUTR 2 Top PJK 1i Bottom PJK 3 Bottom PJK 3 Top