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Microbiology of Sulfate-Reducing Passive Treatment Systems

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

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Microbiology of Sulfate-Reducing Passive Treatment Systems

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  1. Microbiology of Sulfate-Reducing Passive Treatment Systems Amy Pruden2, Sage R. Hiibel1, Luciana P. Pereyra2, Laura Y. Inmann1, Nella Kashani1, Kenneth F. Reardon1, and David Reisman3 1Chemical and Biological Engineering Department, Colorado State University 2Civil and Environmental Engineering Department, Colorado State University 3U.S. Environmental Protection Agency

  2. What is Mining-Influenced Water (MIW)? 4Fe2+(aq) + 8SO42-(aq) + 8H+(aq) When water discharges to surface: 4 Fe+2 + O2 + 10 H2O  4 Fe(OH)3(S) + 8 H+ Yellow Boy

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

  4. H2 CO2 Conceptual SR-PRZ Microbial Community Enzymatic hydrolysis CH4 CO2 Methanogenesis Sulfate reduction HCO3- H2S Fermentation

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

  6. Field Sites

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

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

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

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

  13. ResultsCloning: Diversity Comparison (June)

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

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

  16. PJK LUTR LUTR 2 Top LUTR Top LUTR Bottom PJK 1i Bottom PJK 3 Bottom PJK 3 Top ResultsQPCR: 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. THANK YOU QUESTIONS?

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

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