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Prokaryotic Community Structure and Activity of Sulfate Reducers in Production Water from High-Temperature Oil Reservoirs with and Without Nitrate Treatment

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Prokaryotic Community Structure and Activity of Sulfate Reducers in Production Water from High-Temperature Oil Reservoirs with and Without Nitrate Treatment Prokaryotic community structure and activity of sulfate reducers in production water from high-temperature oil reservoirs with and without nitrate treatment Dr. Antje Gittel Dept. of Biological Sciences, Aarhus University ISMOS 2 Aarhus 2009 Introduction Hypothesis Results Conclusions Offshore oil production systems Separation Oil Gas Water Production Injection ~80 degC 50-60 degC Reservoir ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Characteristics of the study sites Norway Halfdan • Injection of sulfate-rich seawater Denmark • High loads of organic carbon compounds in the reservoir Dan Enrichment of sulfate -reducing prokaryotes Danish Underground Consortium, Maersk Oil (SRP ) that produce H 2S Souring, plugging, biocorrosion, toxicity, ..... • Strategy to control SRP activity at Halfdan: Addition of nitrate to the injection water ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Principles of SRP inhibition by nitrate addition (i) Stimulation of heterothrophic nitrate- reducing bacteria (hNRB ) that hNRB outcompete SRP for electron donors (ii) Activity of nitrate -reducing, sulfide - oxidizing bacteria (NR-SOB ) resulting in SRP NR-SOB a decreasing net production of H2S (iii) Increase in redox potential by the production of nitrite and nitrous oxides and thereby and inhibition of SRP ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Compared to an untreated oil field (Dan), nitrate addition to the injection water at Halfdan stimulates the growth of hNRB and/or NR-SOB , resulting in: 1) A general change in the prokaryotic community composition, i.e. • Presence of competitive hNRB and NR-SOB and/or • Presence of SRP that are able to reduce nitrate instead of sulfate 2) A decrease in SRP abundance and activity and 3) A reduced net sulfide production. ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Prokaryotic community structure and diversity Quality & Quantity Identity & Abundance 16S rRNA & dsrAB gene analyses ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Prokaryotic community structure and diversity Novelty MPN, FISH Quality & Quantity Quantitative PCR (Talk by Ketil Sørensen) Identity & Abundance 16S rRNA & dsrAB gene analyses ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Prokaryotic community structure and diversity Bacterial 16S rRNA genes Dominance of the Firmicutes Dan (- nitrate) Sulfate reducers represented by different genera Deltaproteobacteria Desulfotomaculum spp. Halfdan Nitrate reducers only present at (+ nitrate) Halfdan Epsilonproteobacteria Deferribacterales ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Prokaryotic community structure and diversity Archaeal 16S rRNA genes Dominance of the Thermococcales Dan (- nitrate) Sulfate reducers Archaeoglobus spp . Lower clone frequency at Halfdan Halfdan Nitrate reducers (+ nitrate) (e.g. Pyrobaculum sp., Aeropyrum sp.) were not detected ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Prokaryotic community structure and diversity Bacterial and archaeal dsrAB genes Dan (- nitrate) Deltaproteobacteria Desulfotomaculum spp. Archaeoglobus spp . Same phylotypes as detected via Halfdan (+ nitrate) 16S rRNA-based analyses, but more detailed picture Lower clone frequency of Archaeoglobus relatives at Halfdan ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Prokaryotic community structure and diversity Dan, - nitrate Halfdan, + nitrate Bacteria DIVERSITY Bacteria Archaea Archaea ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Prokaryotic community structure and diversity Total prokaryotes (Quantification of bacterial and archaeal 16S rRNA gene copies with qPCR) (%) Dan 58 (- nitrate) 4.7 x 10 4 gene copies /mL 42 Halfdan 25 (+ nitrate) 1.1 x 10 6 gene copies /mL 75 Archaeal origin Bacterial origin ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Prokaryotic community structure and diversity Total sulfate-reducing prokaryotes (Quantification of deltaproteobacterial and archaeal dsrAB gene copies with qPCR) (% dsrAB / 16S rRNA) Dan 14.1 (- nitrate) 0.7 x 10 4 gene copies /mL 0.4 Halfdan 2.9 (+ nitrate) 3.5 x 10 4 gene copies /mL 0.3 Archaeal origin Bacterial origin ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Prokaryotic community structure and diversity Higher absolute abundances of total prokaryotes and SRP, but less diverse communities at the nitrate-treated field Bacterial sulfate reducers were present in low numbers in both systems, but represented by different genera Archaeal sulfate reducers were highly abundant and dominated the sulfate-reducing community, but their relative abundance was lower in the nitrate-treated field Nitrate reducers (almost) exclusively present in the nitrate-treated system and affilated with bacterial hNRB and NR-SOB ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Activity and identity of SRP in enrichment cultures Bacterial sulfate reduction Archaeal sulfate reduction (60 degC, piping) (80 degC, reservoir) ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Activity and identity of SRP in enrichment cultures Bacterial sulfate reduction Archaeal sulfate reduction (60 degC, piping) (80 degC, reservoir) • No activity in any of the samples ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Activity and identity of SRP in enrichment cultures Bacterial sulfate reduction Archaeal sulfate reduction (60 degC, piping) (80 degC, reservoir) • No activity in any of the samples • Only detectable in samples from Dan (- nitrate) • Activity accompanied by an increase in bacterial 16S rRNA and dsrAB gene copy numbers • Enriched organisms were related to either sulfate- or sulfur-reducing organisms (based on 16S rRNA gene analysis) ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Activity and identity of SRP in enrichment cultures Desulfovibrio vulgaris (U16723) Desulfomicrobium norvegicum (AB061532) dsrAB gene Desulfonatronovibrio hydrogenovorans (AF418197) Desulfobacula toluolica (AJ457136) Production water Desulfobacula phenolica (AF551758) Desulfobacter postgatei (AF418198) Enrichments Desulfobacterium autotrophicum (AF418182) Desulfosarcina variabilis (AF191907) Desulfotomaculum geothermicum (AF273029) Desulfo- Hda_dsr92T (FN376502) [3/31] * tomaculum Desulfobacterium anilini (AF482455) geothermicum Desulfotalea arctica (AY626032) Desulfotalea psychrophila LSv54 (NC_006138) Desulfobulbus elongatus (AJ310430) Desulfobulbus propionicus (AF218452) Desulfobulbus rhabdoformis (AJ250473) Dan_dsr25T (FN376469) [2/29] Dan60S_dsr17E (FN376523) [30/30]* Dan60_dsr11E (FN376523) [30/30]* Syntropho- Hda_dsr20T (FN376491) [5/31] * Desulfacinum infernum (AF418194) bacteraceae Thermodesulforhabdus norvegica (AF334597) Desulfovirga adipica (AF334591) Desulfotomaculum halophilum (AY626024) Archaeoglobus fulgidus (M95624) Dan_dsr69 (FN376461) [27/29]* Archaeo- Hda_dsr24 (FN376483) [23/29] * Archaeoglobus profundus (AF071499) globales Thermodesulfovibrio islandicus (AF334599) Thermodesulfovibrio yellowstonii (U58122) 0.10 ISMOS 2, June 2009 Antje Gittel Introduction Hypothesis Results Conclusions Majority of the obtained sequences from both production systems affiliated with thermophilic prokaryotes indigenous communities responsible for sulfide production Potential effects of nitrate addition at Halfdan 1) General community shift including the appearance of putative nitrate reducers (especially hNRB of the Deferribacterales ) 2) Lower relative abundance of archaeal SRP (Archaeoglobus spp .) 3) Lack of detectable SRP activity 4) Stimulation of the total prokaryotic community indicated by higher gene copies of all genes detected Larger biomass studies needed to estimate the potential of plugging and resumption of sulfide ISMOS 2, June 2009 Antje Gittel Acknowledgements Co-authors of this talk Kjeld Ingvorsen Andreas Schramm Ketil Sørensen Torben Lund Skovhus Technicians at the Microbiology Department, Aarhus University Britta Poulsen Trine Søgaard Thomsen Tove Wiegers Jan Larsen (Maersk Oil, Copenhagen) Adam Wieczorek (University of Bayreuth) .... and you for your attention! ISMOS 2, June 2009 Antje Gittel.
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