Microbial genomics for de-risking offshore oil and gas exploration in Nova Scotia. Casey Hubert

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1 Microbial genomics for de-risking offshore oil and gas exploration in Nova Scotia Casey Hubert

2 Research Funding Geomicrobiology Group marine oil spill bioremediation establishing environmental baselines seabed hydrocarbon seep microbiology deep biosphere microbial ecology extremophiles bacterial endospores oil reservoir microbiology food microbiology (coffee, sourdough)

3 Outline 1. original observations and interest in hydrocarbon seeps 2. using cold-adapted seabed microbes for prospecting 3. using dormant thermophilic bacterial spores for prospecting

4 Smeerenburgfjorden, Svalbard, 80 North

5 sulphate reduction rate (nmol cm -3 h -1 ) SO 4-2 organics SRB H 2 S CO 2 Svalbard marine sediment incubated from -2 to +40 C 600 Bo Barker Jørgensen, unpublished Temperature ( C)

6 sulfate reduction rate (% of optimum) Thermophilic Bacteria in Arctic Sediments Svalbard ~0 C in-situ Hubert et al 2009 Science 325: 1541 sulfate-reducing bacteria SRB incubation temperature ( C)

7 sulfate reduction rate (% of optimum) Thermophilic Bacteria in Arctic Sediments Svalbard ~0 C in-situ Hubert et al 2009 Science 325: 1541 sulfate-reducing bacteria SRB samples pre-pasteurized at 80 C incubation temperature ( C)

8 Desulfotomaculum endospores genus Desulfotomaculum sulfate reducers endospore formers thermophiles SO 4-2 H 2 S SRB simple organics CO 2 sulfate reduction Flemming Mønsted Christensen, M.Sc. thesis, 2009

9 sediment depth (cm) sediment deposition (year) Desulfotomaculum endospores in Arctic marine sediment estimated abundance (thermophiles cm -3 ) Flemming Mønsted Christensen, M.Sc. thesis, per gram of sediment annual influx 10 8 m -2 where are they coming from?

10 Thermospores where are they coming from? rrna gene phylogeny: 5% oil reservoir mid-ocean ridge Caloranaerobacter (2) Caminicella Desulfotomaculum warm anoxic fluid flow

11 seabed fluid flow ~ an unappreciated microbial dispersal vector? bottom water current sedimentation petroleum reservoir Hubert & Judd 2010 Handbook of Hydrocarbon and Lipid Microbiology (Springer) Johnson et al. (2006) Geofluids 6: Hydrocarbon seeps widespread in the ocean fluid seepage from depth depth influences in situ T thermospores have many rrna gene database hits to oil fields Hydrothermal circulation worldwide network of ocean ridges lateral (axis) influence on in situ T thermospores have some rrna gene database hits to mid ocean ridge systems

12 Thermospores where do they come from? seabed fluid flow ~ an unappreciated microbial dispersal vector? rrna gene phylogeny: 5% oil reservoir Caloranaerobacter (2) mid-ocean ridge Caminicella Desulfotomaculum warm anoxic fluid flow

13 Microbiology-based prospecting for seabed hydrocarbon seeps bottom water current sedimentation oil reservoir

14 Microbiology-based prospecting for seabed hydrocarbon seeps bottom water current sedimentation oil reservoir

15 Microbiology-based prospecting for seabed hydrocarbon seeps bottom water current sedimentation O 2 conc. oil reservoir

16 Hypothesis: thermophilic spores [ ] and cold-adapted oil-degrading bacteria [ ] have different distributions at seeps bottom water current sedimentation oil reservoir detectable range?

17 Prospecting advantage using dormant bacterial spores compared to active bacteria, or hydrocarbons? bottom water current sedimentation oil reservoir

18 Prospecting advantage using dormant bacterial spores compared to active bacteria, or hydrocarbons? bottom water current sedimentation oil reservoir

19 Multiple approaches to exploration risk assessment bottom water current sedimentation oil reservoir

20 Outline 1. original observations and interest in hydrocarbon seeps 2. using cold-adapted seabed microbes for prospecting 3. using dormant thermophilic bacterial spores for prospecting

21 Scotian Slope, m Eastern Gulf of Mexico m

22 Nova Scotia Department of Energy s Play Fairway Analysis $15M investment by NS interdisciplinary geoscience mapping >$2B in exploration commitments (Shell, BP, Statoil) Front-end science to characterize the deep seabed reduced risk

23 Canadian Coast Guard Ship: Hudson summer 2015 & 2016

24 Scotian Slope Piston Coring >60 sites sampled 1750 to 3450 m water depth 0 to 10 mbsf sediment depth Geochemical analyses (APT) Organic carbon content Gas wetness Isotopes (δ 13 C, δd) Sulfate depth profiles (UofC) Microbial analysis >400 samples bacterial community composition

25 Depth (cm) Distinct sulfate profiles for Sites M, J, E Sulfate (mm) 0 20 Hydrocarbons = sulfate reduction sulfate-reducing bacteria SRB Site M Site J Site E Site N Site H Geochem (APT) Oye Adebayo

26 Depth (cm) Surface and Subsurface samples for DNA Sulfate (mm) 0 20 Surface: 0 cm & 20 cm (trigger weight core and piston core) Subsurface: >20 cm (only piston core)

27 subsurface (>20 cm) Atribacteria at 14 sites Relative abundance (%) Carmen Li Aerophobetes Aminicenantes Atribacteria Atribacteria Atribacteria Atribacteria Deidra Stacey 7 dominant OTUs OTU_9 OTU_58 OTU_4984 OTU_1 0 A B C D E F H I J K L M N O Atribacteria OTU_ sulfate (U of C) geochem (APT)

28 subsurface (<20 cm) Atribacteria at 15 sites Relative abundance (%) A B C D E F G H I J K L M N O Carmen Li 7 dominant OTUs Chloroflexi Acidobacteria Gammaproteobacteria Alphaproteobacteria Gammaproteobacteria Aminicenantes Atribacteria Deidra Stacey OTU_2 sulfate (U of C) geochem (APT)

29 subsurface (<20 cm) Atribacteria at 15 sites Relative abundance (%) Carmen Li Chloroflexi Acidobacteria Deidra Stacey 7 dominant OTUs Gammaproteobacteria Alphaproteobacteria 5 Gammaproteobacteria Aminicenantes 0 A B C D E F H G I J K L M N O Atribacteria OTU_ sulfate (U of C) geochem (APT)

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34 What is the functional role of uncultivated Atribacteria? Metagenomics N O O OG N N 3 metagenomes site 26 (O) site 29 (OG) site 44 (N) Xiyang Dong 58 draft genomes from three samples 6 Atribacteria preliminary evidence reveals hydrocarbon degradation genes in 5/6 Atribacteria

35 Outline 1. original observations and interest in hydrocarbon seeps 2. using cold-adapted seabed microbes for prospecting 3. using dormant thermophilic bacterial spores for prospecting seep-associated Atribacteria in both Nova Scotia and Gulf of Mexico sediments may be gas or oil associated, and may be capable of hydrocarbon biodegradation ongoing detailed analysis of genomic and oil geochemistry datasets

36 And! using genomics for exploration automatically provides an environmental baseline optimizing conditions for baseline data collection Deidra Stacey 2 Nova Scotia cores triplicate sediment aliquots 3 DNA extraction protocoss 3 PCR primer pairs

37 Outline 1. original observations and interest in hydrocarbon seeps 2. using cold-adapted seabed microbes for prospecting 3. using dormant thermophilic bacterial spores for prospecting

38 Thermospores where do they come from? Are spore-forming Firmicutes really prevalent in oil reservoirs? rrna gene phylogeny: 5% Caloranaerobacter (2) Caminicella Desulfotomaculum warm anoxic fluid flow

39 Are spore-forming Firmicutes really prevalent in oil reservoirs? ~75 studies with amplicon libraries from oil reservoirs only 12 from formation water (no water injection) therefore truly indigenous communities analysed Firmicutes indeed prevalent next step: expand the study to include produced water samples as well as formation waters Daniel Gittins

40 111 surface sediments heated to 50 C Anirban Chakraborty

41 71 oil-positive and 40 oil-negative

42 sulfate reduction rate (nmol cm -3 h -1 ) (% of optimum) Thermophilic Bacteria in cold sediments Svalbard ~0 C in-situ Hubert et al 2009 Science 325: 1541 sulfate-reducing bacteria SRB incubation temperature ( C) 50 C DNA sequencing to characterize the microbial community after several days at 50 C incubation time (hours at 50 o C) 1-30 thermospore OTUs per sediment

43 Thermospore OTUs 115 thermospores 50 C site occupancy

44 top 12 oil-associated thermospores in EGoM 80% detection in oil-positive locations oil-positive oil-negative

45 top 12 oil-associated thermospores in EGoM 80% detection in oil-positive locations oil-positive oil-negative

46 top 12 oil-associated thermospores in EGoM 80% detection in oil-positive locations oil-negative

47 Outline 1. original observations and interest in hydrocarbon seeps 2. using cold-adapted seabed microbes for prospecting 3. using dormant thermophilic bacterial spores for prospecting Conclusion: mutually exclusive strategies for seep detection are showing promising results

48 other applications? thermospores as messengers from the reservoir? bottom water current sedimentation oil reservoir