The prevalence of thiosulfate-reducing fermentative bacteria in oil production facilities Annette T. De Capite, Kathleen E. Duncan, Ralph S. Tanner University of Oklahoma Dept. of Microbiology and Plant Biology Why Consider Thiosulfate Reducing Bacteria? http://voyagesphotosmanu.com/prudhoe_bay_oil.html Sulfidogenesis in the Pipeline Environment 2- Dissimilatory Sulfate SO4 Reduction Pathway Sulfate HS- 2- S2O3 Multiple pathways Thiosulfate Sources of Sulfate and Thiosulfate Sulfate - 2- • Production waters 2 HS S O 2 3 • Seawater 2 O2 H2O • Interconversion to other sulfur anions Thiosulfate 2- 2- SO4 S2O3 • Production waters (Mineral) • Seawater • Oxidation of 2- 2- SO4 S2O3 - sulfides (Seawater) HS Materials Sample [Cl-] (Molar) Temperature Angola production waters 1.5 49oC European production waters 2.5 37oC Middle East Field production waters 2.5 49oC Alaskan North Slope PIG Envelope 0.34 49oC High Temperature, High Salt Production Waters Isolation Results Halanaerobiales isolates obtained from 3 sites! 69% belong to Family Halanerobiales 35 Isolates Total! Screening Isolates for Thiosulfate Reduction Fermentative Sterile Fermentative (No S2O3) (No S2O3) Sterile Thiosulfate-reducing Thiosulfate-reducing (10 mM S2O3) (10 mM S2O3) Confirmation of thiosulfate reduction with terminal electron acceptor assay. Sulfide Producing Isolates Anaerobaculum TR Halanaerobium SP TR: Confirmation of thiosulfate reduction using Halanaerobium SP / TR TEA assay ZB2A SP: Sulfide OKU7 Halanaerobiales SP / TR production when amended with Petrotoga SP thiosulfate Molecular Techniques Middle East Field Anaerobic fermenters common to high salt, high temperature oil production waters. Middle East Field 16S Results (OU Biocorrosion Center) Genus Halanaerobium Family Halanerobiales Phylum Firmicutes Obligate halophiles Isolated from saline production waters, brine lakes, and microbial mats Fermentative metabolism Do not reduce sulfate! 3 species can reduce thiosulfate Bhupathiraju et al 1999 Phylogenetic Relationship of Isolates with Respect to Cultivated Representatives Genus Halanaerobium Novel Halanaerobium ZB2A (Middle East Isolate) TR Species Novel Halanaerobiales OKU7 (Angola Isolate) TR Genus ZB2A: Novel Halanaerobium Species Temperature: 22-50oC, 50oC Salinity: 5-30%, 15% pH: 6.0-9.0, 6.5 Can reduce thiosulfate and elemental sulfur OKU7: Novel Halanaerobiales Genus Temperature: 37-60oC, 45oC Salinity: 1-9%, 7% pH: 6.0-7.5, 6.5 Can reduce thiosulfate, sulfite, and elemental sulfur OKU7: Sulfidogenesis from Sulfur Compounds 6.0 Sulfide produced under thiosulfate, 5.0 sulfite, and sulfur reducing conditions! 4.0 Equivalent sulfide produced under 3.0 fermentative and sulfate reducing conditions. 2.0 Sulfide Conc. (mM) 1.0 0.0 Thiosulfate Sulfate Sulfite Sulfur Fermentative Role of Halanaerobium in Biocorrosion • Corrosive fermentation 2- products S2O3 CO2 and Acetate Volatile fatty acids Oxic • Sulfide produced from Conditions thiosulfate reduction • Thiosulfate can be regenerated abiotically. HS- Ivanova et al 2011 Target Genes for Sulfate Reduction Direct Reduction SO 2- * 2- - 4 APS SO3 HS Sulfate Sulfite * * Sulfide SRB Target Genes: = APS (Adenosine-5’-phosphosulfate) * Reductase = DSR (Dissimilatory sulfite reductase) * Rabus et al 2006 Potential Target Genes for Thiosulfate Reduction Thiosulfate reductase SO 2- + - - 2- 3 H + 2e 2- CN SO3 HS- S2O3 SCN- * Rhodanese-like proteins Thiosulfate H2O Disproportionation 2- + SO4 + H HS- Take home message • SRB are not the only outlaws! • TRB > SRB in some Halanaerobium pipelines. • TRB specific detection is needed. • How do TRB produce sulfide? • Connect enzymatic mechanisms Guilty of sulfide production! to gene targets Suspected of corrosive crimes! Acknowledgements Thesis Committee: Special Thanks: Dr. Neil Wofford Dr. Kathleen Duncan Dr. Athenia Oldham Dr. Ralph Tanner Dr. Deniz Aktas Dr. Joseph Suflita Dr. Tiffany Lenhart Dr. Michael McInerney Chris Marks Renxing Liang Funded by Katy Brown OU Biocorrosion Vince Sandifer Center Thank you for your time. I welcome your questions and comments! .