The prevalence of thiosulfate-reducing fermentative ... prevalence of thiosulfate-reducing fermentative bacteria in oil ... OU Biocorrosion ... The prevalence of thiosulfate-reducing

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

SO4

2-

Sulfate

HS-

Dissimilatory Sulfate

Reduction Pathway

S2O

3

2-

Thiosulfate Multiple pathways

Sources of Sulfate and Thiosulfate

Thiosulfate • Production waters

• Seawater

• Oxidation of sulfides

Sulfate • Production waters

• Seawater

• Interconversion to other sulfur anions

S2O

3

2-

S2O

3

2-

HS-

2 HS- S2O3

2-

2 O2 H2O

SO4

2-

SO4

2-

(Mineral)

(Seawater)

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

Materials

High Temperature, High Salt Production Waters

Isolation Results

35 Isolates Total!

69% belong to Family

Halanerobiales

Halanaerobiales

isolates obtained

from 3 sites!

Screening Isolates for Thiosulfate Reduction

Confirmation of thiosulfate reduction with terminal electron acceptor assay.

Fermentative

(No S2O3)

Thiosulfate-reducing

(10 mM S2O3)

Sterile Fermentative

(No S2O3)

Sterile Thiosulfate-reducing

(10 mM S2O3)

Sulfide Producing Isolates

Halanaerobium SP

Halanaerobium SP

Halanaerobiales SP

Petrotoga SP

TR:

Confirmation of

thiosulfate

reduction using

TEA assay

SP:

Sulfide

production when

amended with

thiosulfate

ZB2A

OKU7

Anaerobaculum TR

/ TR

/ TR

Molecular Techniques

Middle East Field 16S Results (OU Biocorrosion Center)

Anaerobic fermenters common to high salt, high

temperature oil production

waters.

Middle East Field

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 Halanaerobiales

Genus

Novel

Halanaerobium

Species ZB2A (Middle East Isolate) TR

OKU7 (Angola Isolate) TR

ZB2A: Novel Halanaerobium Species

Temperature: 22-50oC, 50oC

Salinity: 5-30%, 15%

Can reduce thiosulfate

and elemental sulfur

pH: 6.0-9.0, 6.5

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

0.0

1.0

2.0

3.0

4.0

5.0

6.0

Thiosulfate Sulfate Sulfite Sulfur Fermentative

Su

lfid

e C

onc. (m

M)

OKU7: Sulfidogenesis from Sulfur Compounds

Sulfide produced under thiosulfate,

sulfite, and sulfur reducing conditions!

Equivalent sulfide produced under

fermentative and sulfate reducing

conditions.

Role of Halanaerobium in Biocorrosion

Ivanova et al 2011

S2O

3

2-

• Sulfide produced from thiosulfate reduction

HS-

•Corrosive fermentation products

CO2 and Acetate

Volatile fatty acids

• Thiosulfate can be regenerated abiotically.

Oxic

Conditions

APS*

Rabus et al 2006

Target Genes for Sulfate Reduction

* *

* *

= APS (Adenosine-5’-phosphosulfate)

Reductase

= DSR (Dissimilatory sulfite reductase)

SRB Target Genes:

SO4

2-

Sulfate

SO3

2-

Sulfite

HS-

Direct Reduction

Sulfide

SO3

2- -CN

SCN-

* Rhodanese-like proteins

S2O

3

2-

+ H+

HS-

SO4

2-

H2O Thiosulfate

Disproportionation

Potential Target Genes for Thiosulfate Reduction

SO3

2-

H+ + 2e

-

HS-

Thiosulfate reductase

Guilty of sulfide production!

Suspected of corrosive crimes!

Halanaerobium

Take home message

•SRB are not the only outlaws!

•TRB > SRB in some pipelines.

•TRB specific detection is needed.

•How do TRB produce sulfide? • Connect enzymatic mechanisms

to gene targets

Thesis Committee:

Dr. Kathleen Duncan

Dr. Ralph Tanner

Dr. Joseph Suflita

Dr. Michael McInerney

Special Thanks:

Dr. Neil Wofford

Dr. Athenia Oldham

Dr. Deniz Aktas

Dr. Tiffany Lenhart

Chris Marks

Renxing Liang

Katy Brown

Vince Sandifer

Acknowledgements

Funded by

OU Biocorrosion Center

Thank you for your time.

I welcome your questions and comments!