Department of Microbiology, University of Oklahoma Investigating Pathways of Sulfidogenesis in Thiosulfate-Reducing Halanaerobium Species Corrosion of hydrocarbon pipelines and production facilities continues to be a major concern for the oil industry. Corrosion occurs both abiotically and biotically, but this afternoon I will be focused on the biotic corrosion induced by microorganisms living within pipelines (MIC). Sulfide production from the reduction of sulfur species is a major factor contributing to MIC. Annette De Capite Department of Microbiology, University of Oklahoma

Sulfidogenic Bacteria Sulfate-reducing bacteria Sulfate (SO42-) Sulfite (SO32-) Sulfide HS- (aq) / H2S (g) Sulfur-reducing bacteria Elemental sulfur (S8) Thiosulfate-reducing bacteria Thiosulfate (S2O32-)

Observations SRB? 1.5-4 M Cl-, 49oC Halanaerobium! Middle East Field site is a high salt, high temperature oil production facility experiencing severe corrosion. Illumina sequencing results of production waters from this site reveal that Halanaerobium compromise a dominant portion of the microbial community in most samples. The high salt, high temperature conditions of the production waters may pose selective pressures which greatly limits the microbial diversity. Other selective pressures to consider: availability of substrates, nutrients, and terminal electrons acceptors. A general trend that has been observed in these high salt, high temperature production facilities are the predominance of anaerobic fermentative microorganisms, so it makes sense to investigate the role (if any) that these anaerobic fermenters may play in biocorrosion. Anaerobic fermenters common to high salt, high temperature oil production waters. Middle East Field 16S Results (OU Biocorrosion Center)

Materials High Temperature, High Salt Production Waters Sample [Cl-] (Molar) Temperature Angola production water 1.5 49oC Middle East Field production water 2.5 European Oil Reservoir 37oC Production waters are water that comes out of oil/gas formation along with the unrefined hydrocarbons. A PIG envelope is a same of the inner scrappings of a pipeline, which is typically composed of corrosion products. High Temperature, High Salt Production Waters Enrichments established under fermentative and thiosulfate-reducing conditions.

23 sulfidogenic isolates obtained! Halanaerobiaceae Isolation Results Genus Halanaerobium Family Halanaerobiaceae 23 sulfidogenic isolates obtained! Emphasize purchased strains. H. congolense, H. kusherni, and H. salsuginis were also obtained from DSM or Dr. McInerney. Novel Genus? TR denotes species confirmed for thiosulfate reduction Bold strains represent species used in this study

Genus Halanaerobium Firmicutes Obligate Anaerobes Obligate halophiles Isolated from saline production waters, brine lakes, and microbial mats Fermentative metabolism Do not reduce sulfate! 4 species produce sulfide from the reduction of thiosulfate and sulfur. 1 µm Ravot et al 2005. Research in Microbiology. Bhupathiraju et al 1999. International Journal of Systematic Bacteriology. Bar = 1 um Gram reaction negative, but cells actually have gram positive cell wall structure. Gram reaction negative because of reduced amount of peptidoglycan in cell wall. Slow down on environments they have been isolates from. Emphasize that this picture is of a Halanaerobium kushneri, a species of Halanaerobium that was isolated from production waters in OK and characterized by the Tanner and McInerney labs. Fermentative metabolism. Sugars and aminoacids serve as electron donor for fermentation. End products vary by species. All species product acetate, carbon dioxide, and hydrogen. Lactate, ethanol, propionate, and butyrate can be produced in different ratios. Rexning has demonstrated that one isolate (obtained from Texas production waters) produces predominantly acetate as a end product of fermentation under thiosulfate-reducing conditions. Halanaerobium kushneri

Sources of Sulfate and Thiosulfate Production waters Seawater Interconversion to other sulfur anions HS- S2O32- SO42- SO42- (Mineral) (Seawater) S2O32- Thiosulfate Production waters Seawater Oxidation of sulfides Sulfate can be present in production waters because some geological formations contain gypsum (CaSO4 2H20), anhydrite gypsum (CaSO4). Sulfate is also present in seawater, so if seawater is employed for flooding hydrocarbon formations or pipelines, sulfate will be introduced to the system. Interconversion of sulfate to thiosulfate (and a variety of other sulfur species) will occur wherever sulfate is present due to both abiotic and biotic reactions. Additionally, in the presence of oxygen, sulfide undergoes ABIOTIC oxidation to thiosulfate. Sulfides are common to minerals and are also the product of sulfate reduction. Oxygen can be introduced into hydrocarbon processing systems if (1) production water is reused that has been exposed to atmosphere oxygen levels or if seawater contains oxygen or (2) if pipelines systems are exposed to atmospheric oxygen during cleaning/maintaince. Emphasize that thiosulfate in pipelines may drive the microbial ecology more than sulfate. Transition into next slide: What types of microorganisms are present in highly saline brines and oil production facilities? S2O32- HS-

Molecular Detection of Sulfidogenic Bacteria 16S rRNA gene Functional genes Applications Assess if microbial community has the genetic potential for sulfidogenesis. Caveats Does not confirm viability of bacteria or activity of sulfidogenic enzymes. Emphasize molecular detection must be confirmed with physiology before implementation! Cultivation and physiology!

Gene Targets for SRB SO32- SO42- APS HS- Amplification ≠ Enzymatic Activity dsrABD aprBA SO32- HS- APS Sulfide Sulfite SO42- Sulfate APS Reductase Dissimilatory Sulfite Reductase *

Gene Targets for TRB rdl dsrABD or asrABC S2O32- S2O32- S2O32- aprBA Sulfate Thiosulfate disproportionation Sulfide HS- aprBA sat HS- SO32- Sulfide Thiosulfate Reductase S2O32- Sulfite phs Molybdopterin protein Sulfite SCN Thiocyanate SO32- Rhodanese S2O32- rdl * * Proposed to be involved in thiosulfate-reduction of Halanaerobium species. Examples of organisms that employ each pathway. Thiosulfate disproportionation: Proteobacteria: Desulfovibrio sulfodismutans, Desulfocapsa sulfexigens, Desulfotomaculum thermobenzoicum Thiosulfate reductase: Salmonella typhimurium, Salmomella enterica, Desulfitobacterium hafniense Sulfide HS- dsrABD or asrABC Sulfite Reductases

Investigating Enzymatic Pathways Assess if other sulfur anions are reduced Evaluate rhodanese activity Identify end products of thiosulfate reduction Determine sulfide/thiosulfate ratio Genomic analysis

ZB2A: Sulfidogenesis from Sulfur Anions Sulfidogenesis in H. vreelandii * Sulfide produced from reduction of S2O32- and S8 Thiosulfate reduction also confirmed in H. congolense and OKU7 Explain how you established fermentative, sulfate reducing conditions. Note: Heat killed control. T-test between fermentative and sulfate reducing conditions to assess if significance difference exists. *Significantly different compared to fermentative, Student’s t-test, p-value < 0.05.

Growth of H. vreelandii in the Presence of Sulfur Anions Similar results observed for H.congolense and OKU7

The Effect of Sulfite on Growth of H. vreelandii Growth is improved as sulfite concentration decreases

Investigating Enzymatic Pathways Assess if other sulfur anions are reduced Elemental sulfur and thiosulfate are reduced to sulfide. Sulfite inhibits growth 2. Evaluate rhodanese activity

Rhodanese activity observed in all Halanaerobium strains tested! Rhodanese Activity: Quantification of Thiocyanate Thiocyanate Iron-thiocyanate complex Measure absorbance of supernatant at 470 nm Ferric nitrate reagent Thiocyanate can only be produced from rhodanese! Ravot et al. 2005. Research in Microbiology. Rhodanese activity observed in all Halanaerobium strains tested!

Rhodanese Activity in H. vreelandii S2O32- SO32- SCN CN (Inhibitory) * Fig. 7: Rhodanese activity was confirmed, as thiocyanate is generated when culture is amended with thiosulfate and cyanide. The amount of sulfite quantified in this assay is significantly lower than the amount of expected, suggesting that sulfite is rapidly converted to prevent accumulation of this inhibitory end product. *Thiocyanate is only detected when culture is amended with cyanide.

Investigating Enzymatic Pathways 2. Evaluate rhodanese activity Both sulfidogenic and non-sulfidogenic species of Halanaerobium possess rhodanese activity. Only a fraction of the predicted sulfite is detected in the rhodanese assay. Where does the sulfite go? 3. Identify end products of thiosulfate reduction

Fate of Sulfur Anions S2O32- SCN SO42- SO32- HS- Thiosulfate Sulfide Sulfite SCN Thiocyanate SO42- Rhodanese Sulfate Thiosulfate reductase Sulfite Reductases CN Sulfate was not produced. Sulfide was generated as an end product of thiosulfate reduction Thiocyanate was produced when cyanide was present. Sulfite was not detected in vivo, but detected in low quantities in the rhodanese assay. Thiosulfate disproportionation Thiosulfate disproportionation (Inhibitory) rdl Thiosulfate reductase phs Thiosulfate disproportionation or phs dsr or ‘asr’

Investigating Enzymatic Pathways 3. Identify end products of thiosulfate reduction Thiosulfate disproportionation can be ruled out as an operable pathway. Rhodanese and thiosulfate reductase cannot not be excluded. Sulfite must be metabolized to prevent growth inhibition 4. Determine sulfide/thiosulfate ratio

1 S2O32- 2 HS- Thiosulfate: Sulfide Ratio SCN SO32- Sulfite SCN Thiocyanate Rhodanese Thiosulfate reductase Sulfite Reductases For every molecule of thiosulfate reduced, two molecules of sulfide are generated (2:1). Suggests that both thiosulfate and sulfite reductases are operable. Sulfite as an intermediate. Questions the applicability of the rhodanese reaction. rdl Thiosulfate reductase phs phs dsr or ‘asr’

Investigating Enzymatic Pathways 4. Determine sulfide/thiosulfate ratio 2:1 Both sulfur atoms of thiosulfate are reduced to sulfide Strongly suggests the activity of a sulfite reductase 5. Genomic analysis

Genome Results Genes for dissimilatory sulfite reductase were not retrieved. Anaerobic sulfite reductase Rhodanese H. congolense Molybdopterin protein Molybdopterin protein could potentially function as thiosulfate reductase. Rhodanese H. vreelandii High quality Rhodanese-like proteins are annotated in the genomes of all Halanaerobium species investigated. Anaerobic sulfite reductase subunits are annotated in the genomes of sulfidogenic Halanaerobium species. Thiosulfate reductase genes were not annotated in any genomes; however, a molybdopterin protein with sequence similarity to phsA was detected. Rhodanese H. kushneri

Compiled Results H. congolense H. vreelandii H. kushneri H. salsuginis   H. congolense H. vreelandii H. kushneri H. salsuginis Thiosulfate-reduction Yes Yes? No Rhodanese Activity Sulfide: Thiosulfate Ratio 2 to 1 1 to 1 NA Predicted Genes Rhodanese, sulfite reductase Rhodanese, thiosulfate reductase, sulfite reductase Rhodanese, thiosulfate reductase NT Yes Talk about how 1:1 ratio in H. kushneri can be explained by lack of sulfite reductase.

? 1 S2O32- 2 HS- Investigating Enzymatic Pathways S-X* SO32- Thiosulfate Sulfide Sulfite S-X* Rhodanese Anaerobic Sulfite Reductase 5. Genome Analysis Initial thiosulfate cleavage remains unclear. Can a rhodanese cleave thiosulfate into sulfite and sulfide? Suggests that an ‘anaerobic sulfite reductase’ is involved in the metabolism of sulfite. Thiosulfate reductase rdl phs ? Mention that in Desulfitobacterium, the operon for phs (thiosulfate reductase) contains two rhodanese domains. ‘asr’ * Sulfur accepting molecule (traditionally CN in rhodanese)

SO32- HS- ‘Anaerobic sulfite reductase’ asrABC Also observed in Thermoanaerobacter, Clostridia and Salmonella species May be involved in reducing sulfite to prevent growth inhibition. Is not amplifiable using gene targets for dissimilatory sulfite reductase from SRB. (Inhibitory) Sulfite asrABC Siroheme dependent? HS- Sulfide

Rethinking TRB Gene Targets Rhodanese-like proteins are not practical gene targets for sulfidogenic Halanaerobium species. Anaerobic sulfite reductase is prime target Directly responsible for the production of sulfide. Inhibition of this enzyme be an effective means of control for sulfidogenic Halanaerobium species.

1 S2O32- 2 HS- Take Home Messages SO32- Sulfidogenesis from various sulfur anions and alternative enzymatic pathways must be considered. Anaerobic sulfite reductase may be a more appropriate gene target for sulfidogenic Halanaerobium species. Thiosulfate Rhodanese Thiosulfate reductase rdl phs SO32- Sulfite Mention that in Desulfitobacterium, the operon for phs (thiosulfate reductase) contains two rhodanese domains. Anaerobic Sulfite Reductase 2 HS- ‘asr’ Sulfide

Acknowledgements Thesis Committee: Dr. Kathleen Duncan Dr. Ralph Tanner Dr. Joseph Suflita Dr. Michael McInerney Thank you for your time. I welcome your questions!