The prevalence of thiosulfate-reducing fermentative 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 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 Obligate halophiles Isolated from saline production waters, brine lakes, and microbial mats Fermentative metabolism Do not reduce sulfate! 3 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

SulfideConc. (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!