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
ISMOS2
Aarhus 2009
Introduction
Hypothesis
Results
Conclusions
Offshore oil production systems
Separation
Oil
Gas
Water
Production
Injection
50-60 degC
~80 degC
Reservoir
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Characteristics of the study sites
Conclusions
Norway
Halfdan
• Injection of sulfate-rich seawater
Denmark
• High loads of organic carbon compounds in
the reservoir
Dan
Enrichment of sulfate-reducing prokaryotes
(SRP) that produce H2S
Danish Underground Consortium, Maersk Oil
Souring, plugging, biocorrosion,
toxicity, .....
• Strategy to control SRP activity at Halfdan:
Addition of nitrate to the injection water
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Conclusions
Principles of SRP inhibition by nitrate addition
(i) Stimulation
reducing
of
heterothrophic
bacteria
nitrate-
(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
ISMOS2, 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.
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Conclusions
Prokaryotic community structure and diversity
Quality & Quantity
Identity & Abundance
16S rRNA & dsrAB
gene analyses
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Prokaryotic community structure and diversity
Conclusions
Novelty
MPN, FISH
Quality & Quantity
Quantitative PCR
(Talk by Ketil Sørensen)
Identity & Abundance
16S rRNA & dsrAB
gene analyses
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Conclusions
Prokaryotic community structure and diversity
Bacterial 16S rRNA genes
Dan
(- nitrate)
Dominance of the Firmicutes
Sulfate reducers
different genera
represented
by
Deltaproteobacteria
Desulfotomaculum spp.
Halfdan
(+ nitrate)
Nitrate reducers only present at
Halfdan
Epsilonproteobacteria
Deferribacterales
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Conclusions
Prokaryotic community structure and diversity
Archaeal 16S rRNA genes
Dan
(- nitrate)
Dominance of the Thermococcales
Sulfate reducers
Archaeoglobus spp.
Lower clone frequency at Halfdan
Halfdan
(+ nitrate)
Nitrate reducers
(e.g. Pyrobaculum sp., Aeropyrum sp.)
were not detected
ISMOS2, 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.
Halfdan
(+ nitrate)
Same phylotypes as detected via
16S rRNA-based analyses, but more
detailed picture
Lower
clone
frequency
of
Archaeoglobus relatives at Halfdan
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Conclusions
Prokaryotic community structure and diversity
Dan, - nitrate
Halfdan, + nitrate
Bacteria
DIVERSITY
Bacteria
Archaea
Archaea
ISMOS2, June 2009
Antje Gittel
Hypothesis
Introduction
Conclusions
Results
Prokaryotic community structure and diversity
Total prokaryotes
(Quantification of bacterial and archaeal 16S rRNA gene copies with qPCR)
(%)
Dan
(- nitrate)
4.7 x 104 gene copies /mL
58
42
Halfdan
(+ nitrate)
1.1 x 106 gene copies /mL
25
75
Archaeal origin
Bacterial origin
ISMOS2, 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
(- nitrate)
14.1
0.7 x 104 gene copies /mL
0.4
Halfdan
(+ nitrate)
2.9
3.5 x 104 gene copies /mL
0.3
Archaeal origin
Bacterial origin
ISMOS2, 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
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Conclusions
Activity and identity of SRP in enrichment cultures
Bacterial sulfate reduction
(60 degC, piping)
Archaeal sulfate reduction
(80 degC, reservoir)
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Conclusions
Activity and identity of SRP in enrichment cultures
Bacterial sulfate reduction
(60 degC, piping)
Archaeal sulfate reduction
(80 degC, reservoir)
• No activity in any of the samples
ISMOS2, June 2009
Antje Gittel
Introduction
Hypothesis
Results
Conclusions
Activity and identity of SRP in enrichment cultures
Bacterial sulfate reduction
(60 degC, piping)
Archaeal sulfate reduction
(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)
ISMOS2, 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)
Hda_dsr92T (FN376502) [3/31] *
Desulfobacterium anilini (AF482455)
Desulfotalea arctica (AY626032)
Desulfotomaculum
geothermicum
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]*
Hda_dsr20T (FN376491) [5/31] *
Syntrophobacteraceae
Desulfacinum infernum (AF418194)
Thermodesulforhabdus norvegica (AF334597)
Desulfovirga adipica (AF334591)
Desulfotomaculum halophilum (AY626024)
Archaeoglobus fulgidus (M95624)
Dan_dsr69 (FN376461) [27/29]*
Hda_dsr24 (FN376483) [23/29] *
Archaeoglobales
Archaeoglobus profundus (AF071499)
Thermodesulfovibrio islandicus (AF334599)
Thermodesulfovibrio yellowstonii (U58122)
0.10
ISMOS2, 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
ISMOS2, 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!
ISMOS2, June 2009
Antje Gittel